The objective of this study was to elucidate the mechanisms of mitochondrial H2O2 generation in mouse organs by determining the nature of their differences in substrate utilization, inhibitor sensitivity, and the site specificity affecting H2O2 production. Mitochondria were isolated from heart, brain, and kidney and the rate of H2O2 generation was measured using the FADH-linked substrates succinate and alpha-glycerophosphate as well as the NADH-linked substrates pyruvate/malate, beta-hydroxybutyrate, and glutamate. Respiratory inhibitors, antimycin and rotenone, were added singly and sequentially to each substrate-supported H2O2 generation reaction mixture to determine the mitochondrial site(s) of generation and the optimal condition(s) for maximal rates of generation. Succinate supported the highest rate of mitochondrial H2O2 generation. Moreover, it was the preferred substrate for the heart mitochondria. alpha-Glycerophosphate is a poor substrate for H2O2 generation in heart mitochondria. Inhibitor studies showed that heart mitochondria were the most sensitive and responsive to antimycin, while brain was the most sensitive to rotenone. A surprising finding was that NADH-linked substrate-supported H2O2 generation in kidney mitochondria was not responsive to rotenone. The contribution from each of the three sites (ubiquinone, NADH dehydrogenase, and alpha-glycerophosphate dehydrogenase) of mitochondrial H2O2 generation to the total was both substrate and organ dependent. Results indicate that assay conditions must be considered before comparisons of sites and rates of mitochondrial H2O2 generation among different organs can be made.