In this work, the topology of mitochondrial O2(-)(radical) and H2O2 generation and their interplay with matrix GSH in isolated heart mitochondria were examined. We observed that complex I releases O2(-)(radical) into the matrix (where it is converted to H2O2 by Mn-SOD) but not into the intermembrane space. No free radical generation was observed from complex II, but succinate treatment caused H2O2 generation from the matrix through a reverse electron flow to complex I. Complex III was found to release O2(-)(radical) into the matrix and into the intermembrane space. Antimycin, which increases steady-state levels of UQO>- (ubisemiquinone at the Qo site) in complex III, enhanced both H2O2 generation from the matrix and O2(-)(radical) production from the intermembrane space. On the other hand, myxothiazol, which inhibits UQO>- formation, completely inhibited antimycin induced O2(-)(radical) toward the intermembrane space and inhibited H2O2 generation from the matrix by 70%. However, myxothiazol alone enhanced H2O2 production from complex III, suggesting that other components of complex III besides the UQO- can cause O2(-)(radical) generation toward the matrix. As expected, mitochondrial GSH was found to modulate H2O2 production from the matrix but not O2- generation from the intermembrane space. Low levels of GSH depletion (from 0-40%, depending on the rate of H2O2 production) had no effect on H2O2 diffusion from mitochondria. Once this GSH depletion threshold was reached, GSH loss corresponded to a linear increase in H2O2 production by mitochondria. The impact of 50% mitochondrial GSH depletion, as seen in certain pathological conditions in vivo, on H2O2 production by mitochondria depends on the metabolic state of mitochondria, which governs its rate of H2O2 production. The greater the rate of H2O2 generation the greater the effect 50% GSH depletion had on enhancing H2O2 production.