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The opening scene shows the Umbrella attack on Arcadia. The tiltrotors fire missiles at the ship before deploying soldiers. Many of the Arcadia test subjects are killed in the attack, whether directly shot by Umbrella soldiers or by getting in the way of them and Alice. As the battle continues on, Alice sinks into the ocean.
Another 'Alice' clone wakes up in a peaceful recreation of suburban Raccoon City in 2002. She and her husband, Todd are late for work due to problems with their alarm clock, and their daughter Becky should be ready for school. As they have their breakfast, a group of zombies run into the house and attack Todd. Alice and Becky run into a storeroom to escape from the horde, making their way into the attic through a hole in the ceiling. They make they way down the ladder, keeping quiet to avoid the zombies finding them. As they escape outside, they are picked up by a clone of Rain. Their ride doesn't go very far, unfortunately, when a truck smashes into the car and flips it over. Alice and Becky escape, believing Rain to be dead - when she awakens, she finds that the zombies are ignoring her for now.
Running into a house, the two hide in a nursery closet. When the zombie becomes interested in the closet, Alice opens it and attacks the creature, drawing it away from Becky. As they fight on the landing, Alice succeeds in pushing it over the balcony. Alice is then attacked by the zombified Todd.
Project Alice re-awakens inside Cell 919, part of Umbrella Prime's prison complex. There, she is interrogated by Jill regarding her reason for betraying Umbrella, and what organization she works for. Jill refuses to acknowledge Alice's own questioning of what happened to her, and where Claire is. She is tortured with white noise at random points to drive her to confess. When the facility's security system mysteriously deactivates, Alice dons her battle armor and escapes down a corridor, ending up in a recreation of Tokyo. She fails to get into a Tokyo Metropolitan Police Department (警視庁 Keishichō) cruiser, so smashes the window with a bicycle chain to steal a handgun from the glove compartment.
As Alice readies herself for combat, the Prime computer announces the activation of the Tokyo "sequence". It begins to rain and cloned Japanese civilians begin walking through the streets with their umbrellas out. The clone representing the police cruiser's driver shouts out about the vandalism done to the car window. A clone of J Pop Girl walks through the streets, brushing up against Alice. She attacks the clone of the Japanese business man, the original killed in 2002. The others run away as more zombies appear. Alice gets their attention and runs out of Tokyo and back into the stetile corridor. A dozen zombies get into the corridor, forcing Alice to take them out with her impressive kicks and her skill with chains. J Pop Girl is left as the only surviving zombie. As her squid-like second set of jaws make their appearance, Alice shoots straight through her head. With the dozen attacking zombies dead, Alice is greeted by even more zombified Edokko enter the corridor. She escapes through another door before they can catch her.
Arriving at Umbrella Central Control, Alice is confused to find that all of the Umbrella operatives have been shot in the head, seemingly uninfected. A weapon rack ascends from the floor after the corpses. As she takes a knife from the rack, a red-clad woman confronts her. They begin fighting, but Alice succeeds in putting a knife to her throat. She knows exactly who the woman is - Ada Wong, one of Wesker's top agents. Ada explains that she no longer works for Umbrella, followed by Wesker himself, as he talks to Alice via computer screen.
As Jill and the other Umbrella operatives begin to investigate how Alice escaped, Ada explains that it was she who temporarily shut down the facility and freed her, under orders from Wesker. He explains that he and Alice working together is for the benefit of preventing human extinction, something Umbrella is failing at. 'Umbrella Prime' is revealed to be an old facility designed some time before 2002 and intended to display realistic simulations of viral outbreaks for the benefit of consumers. A simuation of Moscow would be for the Americans; one of New York for the Russians; one of Tokyo for the Chinese and one of Beijing to the Japanese. The facility itself is located underwater in the Arctic Ocean, meaning Alice simply can't walk out the door. To help her, Wesker has sent in a team.
Leon S. Kennedy leads a team through the snow to get to Alice. Securing an elevator, they travel down to meet up with her. Ada reveals that Luthur West is also a member of this team. As Jill Valentine moves through Tokyo, Wesker reveals that she is under the control of the Red Queen, who has taken control over the entire company. Alice is instructed to move through the New York environment and into the "Suburbia" (Raccoon City) simulation, where she will meet up with the strike team. They are then to move into the Moscow environment and to the old Soviet submarine pens and up the elevator. Leon's strike team has already gained control of the submarine pens and are on their way.
As Alice and Ada enter the New York simulation, they are confronter by two agile "Axemen", who they eventually kill by shooting at a taxi's gasoline tank. With the only two enemies defeated, the simulation ends and the lights turn back on. The Red Queen orders the release of the Las Plagas Undead into the Moscow environment to deal with Leon's group. The Undead arrive dressed in military uniforms and shooting at the strike team.
Entering the Suburbia environment, Alice and Ada see that a simulation has come to an end. Walking into a house, she discovers the body of her clone; Ada explains that Alice represented one of the fifty 'basic' models. With only enough memories to guarantee an appropriate emotional response, Ada explains that the basic clone models could be put in various environments and assume an appropriate character (a businesswoman in New York, a mother in Suburbia), and offers the suggestion that Umbrella's own soldiers could be clones. Alice searches upstairs, where she is attacked by a zombie hiding in the nursery. With the zombie gone, Becky runs into the room. Having hid when her "mother" died, Becky hugs Alice, confused as to why she looks so different.
Taking Becky back to her "mother"'s house, Alice looks at the pictures of her clone and Todd. Ada tries to remind her that none of the photographs are real; that Becky could be anyone in a simulation. Walking outside, they are confronted by clones of Rain; Carlos Olivera and One, created as Umbrella soldiers. Ada gives Alice her hookshot and sends her off to the Moscow zone while she distracts the soldiers, escaping herself by dropping into a ventilation shaft.
Leon's team continue to be assaulted by the Russian troops. Having lost much of their makeshift bunker to a rocket launcher, a chainsaw-armed soldier tears through one of the strike team members. Luthur kills it before it can take out Leon.
Alice and Becky move into the Moscow area, finding themselves in a subway station. The friendly Rain clone discovers the two after inadvertently crossing through the holographic background. Confused as to why everything is written in Russian and Alice is wearing an "S&M get-up". While being a gun control activist, Alice forces her to take up arms to protect Becky while she investigates upstairs. Becky mentions Rain's "sister", who isn't particularly nice; this Rain, not programmed with memories of a sister, is left confused.
Having escaped the army through a mall, Leon's team continues on their search for Alice, losing another man - this time to a giant Licker. Alice arrives, crashing right into it with a car. As they drive off, their car is attacked by motorcyclist Undead. They drive around Moscow as a truck follows with RPG fire. The giant Licker returns, knocking over a truck as it charges at Alice's car.
As the team enters the subway, they find Rain and Becky safe. Using a captured display, Alice works out where the zone exit should be. Leon explains that he isn't one of Wesker's agents; a recent change in events has led to him accepting this mission. Moving into the submarine pens, the power goes out. The Red Queen activates a biohazard in this area to prevent their final escape. The giant Licker arrives, knocking Barry to the ground. Throwing Rain at a wall and snapping her neck, it wraps around Becky with its long tongue and takes her up the elevator shaft.
Alice runs back to the vents to rescue her, despite Leon's objections that she isn't important. The "bad" Rain; "One"; Carlos and Jill then arrive. They shoot Luthur as Alice leaves. An injured Barry and Leon continue shooting while awaiting Alice's return. With only five minutes left before the elevator should activate, they have to be there or they won't be rescued. Barry offers to stay behind and watch everyone else get on board.
Alice finds Becky stuck inside some sort of cocoon, with one of Leon's dead friends in another. She begins feeling pain from a gunshot wound sustained at Suburbia.
Ada is captured by Umbrella, and used as a hostage to get Barry to come out. He offers himself up, making a few hits before dying from his wounds.
Alice discovers the room where the mindless clones are transported for tests. Becky becomes distressed when she sees an antire production line of Alices, and experiences an existential crisis. A bomb goes off in the factory room as the time runs out; more and more of Umbrella Prime falls apart, and its environments are washed away in great floods. The elevator begins ascending as Barry's corpse; Carlos and "One" are washed away. It stops a few meters short of the surface, but they make their way up; Alie and Becky greet Leon and Luthur from a ventilation hatch.
Driving over the ice, the team is surprised to find the ice breaking apart, knocking their snomobile over. It is revealed to be a submarine, which Jill and Rain had successfully commandeered with their Ada hostage. Rain injects herself with the Las Plagas parasite, which Leon has some prior experince with. Leon and Luthur shoot her, but she ejects the casings by moving them through to her fingernails. Their fight descends into mere punches and kicks as Alice and Jill continue their fight.
Alice is almost killed by Jill; however, when Jill attempts to throw Alice into the snowmobile's running caterpillars. Jill is able to regain some control of herself and hesitates. Alice kicks off the device on Jill's chest which is controlling her, and destroys it. Jill goes back to normal and passes out. Rain kills Luther with a superhuman punch which fatally brakes his ribcage, crushing his heart.
Rain then attempts to kill Alice with a similar attack, but she fails to die instantly. Recovering, Alice discovers zombies have escaped the facility and are trying to dig through the ice. Jill gets up and throws Alice a machine gun. Rain yells "You can't kill me!". Alice shoots the ice under her, and she is dragged down and eaten by the zombies. She shakes them loose enough to shout at Alice, only to be dragged down by more.
Alice and the other survivors are then rescued by Wesker's team and get taken to the White House, being used as a resistance base by the few remaining military personnel. Wesker, who has taken to sitting in the vacant Oval Office, infects Alice with the T-virus again so that she can regain her lost powers in order to fight the Red Queen. Alice, Jill, Leon, and Ada follow Wesker to the roof and see it is indeed the last stand, as the millions of zombies and giant Lickers breach the perimeter surrounding the White House, and Kipepeo begin their attack.