We have previously reported that aspirin induces apoptosis in manganese superoxide dismutase (MnSOD)-deficient Saccharomyces cerevisiae cells when cultivated on the non-fermentable carbon source ethanol. Here, we investigated the role of mitochondria in aspirin-induced apoptosis. We report that aspirin had an inhibitory effect on cellular respiration, and caused the release of most of the mitochondrial cytochrome c and a dramatic drop in the mitochondrial membrane potential (DeltaPsi(m)). Also, aspirin reduced the intracellular cytosolic pH in the MnSOD-deficient cells growing in ethanol medium, but this did not seem to be the initial trigger that committed these cells to aspirin-induced apoptosis. Furthermore, loss of DeltaPsi(m) was not required for aspirin-induced release of cytochrome c, since the initial release of cytochrome c occurred prior to the disruption of the DeltaPsi(m). It is thus possible that cytochrome c release does not involve the early onset of the mitochondrial permeability transition, but only an alteration of the permeability of the outer mitochondrial membrane.