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 $$O_2^{\bar .}$$ 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 $$\Delta \tilde \mu _{{\rm H}^ + }$$ -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 (∼80%) proceeds via Complex I at the expense of its $$\Delta \tilde \mu _{{\rm H}^ + }$$ -dependent reduction (reverse electron transfer). At saturating NADH the rate of $$O_2^{\bar .}$$ 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 (∼50 µM)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.