A 16 h exposure of U937 cells to 2.5 μM arsenite promotes superoxide (Symbol) formation and inhibition of the activity of aconitase, a Symbol sensitive enzyme. Both responses were abolished by the complex I inhibitor rotenone, or by the respiration-deficient phenotype. Interestingly, a similar suppressive effect was mediated by a short term pre-exposure to a low concentration of L-ascorbic acid (AA), previously shown to be actively taken up by the cells and by their mitochondria. The mitochondrial origin of Symbol was confirmed by fluorescence microscopy studies, whereas different approaches failed to detect a contribution of NADPH oxidase. Under similar conditions, arsenite induced autophagy as well as a decline in mitochondrial membrane potential resulting in delayed (48 h) apoptosis. Importantly, all these events turned out to be sensitive to treatments associated with prevention of Symbol formation, including AA, and were only partially blunted by inhibitors of autophagy. As a final note, the toxic effects mediated by Symbol were entirely dependent on its conversion to H2O2. AA-sensitive mitochondrial Symbol formation is therefore involved in autophagy and apoptosis induced by arsenite in U937 cells, although part of the lethal response appears mediated by an autophagy-independent mechanism.