In previous studies, we observed that aspirin, a promising cancer-preventive agent, induces apoptosis in mitochondrial manganese superoxide dismutase (MnSOD)-deficient Saccharomyces cerevisiae cells grown aerobically in ethanol medium. In this study, we show that aspirin-induced apoptosis is associated with a significant increase in mitochondrial and cytosolic O2 ·- and oxidation of mitochondrial NAD(P)H. A concomitant rise in the level of cytosolic CuZnSOD activity failed to compensate for mitochondrial MnSOD deficiency. However, an observed increase in activity of Escherichia coli FeSOD targeted to the mitochondrial matrix of the MnSOD-deficient yeast cells, markedly decreased aspirin-induced accumulation of mitochondrial O2 ·-, significantly increased the mitochondrial NAD(P)H level and rescued the apoptotic phenotype. Indeed, recombinant yeast cells expressing E. coli FeSOD behaved in a similar manner to the parent wild-type yeast cells with native mitochondrial MnSOD activity. Wild-type cells consistently showed a decrease in mitochondrial O2 ·- and an increase in mitochondrial NAD(P)H levels in the presence of aspirin in ethanol medium. In fact, in wild-type cells, our studies supported an antioxidant action of aspirin. Taken together, our results indicate that a pro-oxidant effect of aspirin occurring predominantly in cells with compromised mitochondrial redox balance may be enough to overcome antioxidant defences resulting in apoptosis, as observed in MnSOD-deficient yeast cells.