This study investigates the generation of superoxide anion (O₂⁻) by the NADH dehydrogenase in bovine heart mitochondria. Submitochondrial particles from bovine heart mitochondria, when reduced by NADH and rotenone or by reversed electron transfer, produce 0.9 ± 0.1 nmol of O₂⁻ per minute per mg of protein at pH 7.4 and 30°C. The production of O₂⁻ is enhanced by rotenone, antimycin, and cyanide. In NADH- and antimycin-supplemented submitochondrial particles, rotenone has a biphasic effect: it increases O₂⁻ production at the NADH dehydrogenase and inhibits O₂⁻ production at the ubiquinone–cytochrome b site. The generation of O₂⁻ by the NADH dehydrogenase supported by reversed electron transfer is inhibited by rotenone, the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and oligomycin. Cyanide does not affect O₂⁻ generation at the NADH dehydrogenase but inhibits O₂⁻ production at the ubiquinone–cytochrome b site. The production of O₂⁻ at the NADH dehydrogenase is about 50% of that at the ubiquinone–cytochrome b area at pH 7.4. The additivity of the two mitochondrial sites of O₂⁻ generation is observed over the pH range from 7.0 to 8.8. An O₂⁻-dependent autocatalytic process that requires NADH, submitochondrial particles, and adrenaline is described. Isolated mitochondria in State 4 generate 0.6–1.0 nmol of H₂O₂ per minute per mg of protein, which accounts for about 2% of the oxygen uptake under physiological conditions. The effect of specific mitochondrial inhibitors on H₂O₂ production indicates a reduced member of the respiratory chain located between the rotenone- and antimycin-sensitive sites as the main autoxidizable species generating H₂O₂ in a side reaction to the main electron flow. Production of O₂⁻ by mitochondrial membranes in the presence of succinate and antimycin was first reported by Loschen et al. (1971). Subsequently, Boveris & Cadenas (1975) and Dionisi et al. (1975) showed that O₂⁻ can be considered a stoichiometric precursor of mitochondrial H₂O₂. The production of O₂⁻ by isolated succinate–cytochrome c reductase and the effect of protophores and ionophores on H₂O₂ production by coupled mitochondria support the idea that ubisemiquinone is the main univalent reThis study investigates the generation of superoxide anion (O₂⁻) by the NADH dehydrogenase in bovine heart mitochondria. Submitochondrial particles from bovine heart mitochondria, when reduced by NADH and rotenone or by reversed electron transfer, produce 0.9 ± 0.1 nmol of O₂⁻ per minute per mg of protein at pH 7.4 and 30°C. The production of O₂⁻ is enhanced by rotenone, antimycin, and cyanide. In NADH- and antimycin-supplemented submitochondrial particles, rotenone has a biphasic effect: it increases O₂⁻ production at the NADH dehydrogenase and inhibits O₂⁻ production at the ubiquinone–cytochrome b site. The generation of O₂⁻ by the NADH dehydrogenase supported by reversed electron transfer is inhibited by rotenone, the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and oligomycin. Cyanide does not affect O₂⁻ generation at the NADH dehydrogenase but inhibits O₂⁻ production at the ubiquinone–cytochrome b site. The production of O₂⁻ at the NADH dehydrogenase is about 50% of that at the ubiquinone–cytochrome b area at pH 7.4. The additivity of the two mitochondrial sites of O₂⁻ generation is observed over the pH range from 7.0 to 8.8. An O₂⁻-dependent autocatalytic process that requires NADH, submitochondrial particles, and adrenaline is described. Isolated mitochondria in State 4 generate 0.6–1.0 nmol of H₂O₂ per minute per mg of protein, which accounts for about 2% of the oxygen uptake under physiological conditions. The effect of specific mitochondrial inhibitors on H₂O₂ production indicates a reduced member of the respiratory chain located between the rotenone- and antimycin-sensitive sites as the main autoxidizable species generating H₂O₂ in a side reaction to the main electron flow. Production of O₂⁻ by mitochondrial membranes in the presence of succinate and antimycin was first reported by Loschen et al. (1971). Subsequently, Boveris & Cadenas (1975) and Dionisi et al. (1975) showed that O₂⁻ can be considered a stoichiometric precursor of mitochondrial H₂O₂. The production of O₂⁻ by isolated succinate–cytochrome c reductase and the effect of protophores and ionophores on H₂O₂ production by coupled mitochondria support the idea that ubisemiquinone is the main univalent re