The mitochondrial respiratory chain is a major source of reactive oxygen species (ROS) under pathological conditions including myocardial ischemia and reperfusion. Limitation of electron transport by the inhibitor rotenone immediately before ischemia decreases the production of ROS in cardiac myocytes and reduces damage to mitochondria. We asked if ROS generation by intact mitochondria during the oxidation of complex I substrates (glutamate, pyruvate/malate) occurred from complex I or III. ROS production by mitochondria of Sprague-Dawley rat hearts and corresponding submitochondrial particles was studied. ROS were measured as H2O2 using the amplex red assay. In mitochondria oxidizing complex I substrates, rotenone inhibition did not increase H2O2. Oxidation of complex I or II substrates in the presence of antimycin A markedly increased H2O2. Rotenone prevented antimycin A-induced H2O2 production in mitochondria with complex I substrates but not with complex II substrates. Catalase scavenged H2O2. In contrast to intact mitochondria, blockade of complex I with rotenone markedly increased H2O2 production from submitochondrial particles oxidizing the complex I substrate NADH. ROS are produced from complex I by the NADH dehydrogenase located in the matrix side of the inner membrane and are dissipated in mitochondria by matrix antioxidant defense. However, in submitochondrial particles devoid of antioxidant defense ROS from complex I are available for detection. In mitochondria, complex III is the principal site for ROS generation during the oxidation of complex I substrates, and rotenone protects by limiting electron flow into complex III.