Besides major NADH-, succinate-, and other substrate oxidase reactions resulting in four-electron reduction of oxygen to water, the mitochondrial respiratory chain catalyzes one-electron reduction of oxygen to superoxide radical O(2)(-.) followed by formation of hydrogen peroxide. In this paper the superoxide generation by Complex I in tightly coupled bovine heart submitochondrial particles is quantitatively characterized. The rate of superoxide formation during Deltamu(H(+))-controlled respiration with succinate depends linearly on oxygen concentration and contributes approximately 0.4% of the overall oxidase activity at saturating (0.25 mM) oxygen. The major part of one-electron oxygen reduction during succinate oxidation (approximately 80%) proceeds via Complex I at the expense of its Deltamu(H(+))-dependent reduction (reverse electron transfer). At saturating NADH the rate of O(2)(-.) formation is substantially smaller than that with succinate as the substrate. In contrast to NADH oxidase, the rate-substrate concentration dependence for the superoxide production shows a maximum at low (approximately 50 microM) concentrations of NADH. NAD+ and NADH inhibit the succinate-supported superoxide generation. Deactivation of Complex I results in almost complete loss of its NADH-ubiquinone reductase activity and in increase in NADH-dependent superoxide generation. A model is proposed according to which complex I has two redox active nucleotide binding sites. One site (F) serves as an entry for the NADH oxidation and the other one (R) serves as an exit during either the succinate-supported NAD+ reduction or superoxide generation or NADH-ferricyanide reductase reaction.