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Resident Evil 0's main gameplay feature, the "partner zapping" system, is unique in the series in that instead of choosing a single character to play the entire game, the player controls both Rebecca and Billy. Both characters can travel together with one character being controlled by the player and the other by the computer (who can also be partially controlled with the C-stick), or split up and search separate areas each at the same time. This dual control is central to solving some of the puzzles in the game. The difference between both characters is that Rebecca can acquire chemicals with her mixing kit and can get into tight spaces, but is more fragile to enemy attacks. In contrast, Billy can handle heavy objects, has an oil lighter and can sustain more damage than Rebecca, but cannot mix herbs (in contrast from previous games, in which any character could mix herbs).
The game also does away with the use of "item boxes" placed in fixed locations for the player to store items in. Instead, the player can now drop items on the floor to make room for new ones, and must retrace their steps back to the room they previously dropped the discarded item when they need it later in the game. The locations of dropped or "touched" items are displayed on the player's map.