Salicylates and nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in a variety of cancer cells, including those of colon, prostate, breast, and leukemia. We examined the effects of sodium salicylate (NaSal) on reactive oxygen species (ROS) production and the association of these effects with apoptotic tumor cell death. We demonstrate that NaSal mediates ROS production followed by a decrease in mitochondrial membrane potential (deltapsi(m)), release of cytochrome c, and activation of caspase-9 and caspase-3. However, expression of Bcl-2 or Bcl-x(L) prevents ROS production and subsequent loss of deltapsi(m), thereby inhibiting apoptotic cell death. The presence of ROS scavengers and an inhibitor of NADPH oxidase or expression of a dominant negative form of Rac1 blocks ROS production, deltapsi(m) collapse, and the subsequent activation of caspases. These observations indicate that NaSal mediates ROS production critical in the triggering of apoptotic tumor cell death through a Rac1-NADPH oxidase-dependent pathway. Our data collectively imply that NaSal-induced ROS are key mediators of deltapsi(m) collapse, which leads to the release of cytochrome c followed by caspase activation, culminating in tumor apoptosis.