Autophagy is involved in human diseases and is regulated by reactive oxygen species (ROS) including superoxide (O(2)(*-)) and hydrogen peroxide (H(2)O(2)). However, the relative functions of O(2)(*-) and H(2)O(2) in regulating autophagy are unknown. In this study, autophagy was induced by starvation, mitochondrial electron transport inhibitors, and exogenous H(2)O(2). We found that O(2)(*-) was selectively induced by starvation of glucose, L-glutamine, pyruvate, and serum (GP) whereas starvation of amino acids and serum (AA) induced O(2)(*-) and H(2)O(2). Both types of starvation induced autophagy and autophagy was inhibited by overexpression of SOD2 (manganese superoxide dismutase, Mn-SOD), which reduced O(2)(*-) levels but increased H(2)O(2) levels. Starvation-induced autophagy was also inhibited by the addition of catalase, which reduced both O(2)(*-) and H(2)O(2) levels. Starvation of GP or AA also induced cell death that was increased following treatment with autophagy inhibitors 3-methyladenine, and wortamannin. Mitochondrial electron transport chain (mETC) inhibitors in combination with the SOD inhibitor 2-methoxyestradiol (2-ME) increased O(2)(*-) levels, lowered H(2)O(2) levels, and increased autophagy. In contrast to starvation, cell death induced by mETC inhibitors was increased by 2-ME. Finally, adding exogenous H(2)O(2) induced autophagy and increased intracellular O(2)(*-) but failed to increase intracellular H(2)O(2). Taken together, these findings indicate that O(2)(*-) is the major ROS-regulating autophagy.