Interruption of electron flow at the quinone-reducing center (Q(i)) of complex III of the mitochondrial respiratory chain results in superoxide production. Unstable semiquinone bound in quinol-oxidizing center (Q(o)) of complex In: is thought to be the sole source of electrons for oxygen reduction; however, the unambiguous evidence is lacking We investigated the effects of complex III inhibitors antimycin, myxothiazol, and stigmatellin on generation of H2O2 in rat heart and brain mitochondria. In the absence of antimycin A, myxothiazol stimulated H2O2 production by mitochondria oxidizing malate, succinate, or cr-glycerophosphate. Stigmatellin inhibited H2O2 production induced by myxothiazol. Myxothiazol-induced H2O2 production was dependent on the succinate/fumarate ratio but in a manner different from H2O2 generation induced by antimycin A. We conclude that myxothiazol-induced H2O2 originates from a site located in the complex III Q(o) center but different from the site of H2O2 production inducible by antimycin A