The study by Turrens and Boveris investigates the generation of superoxide anion (O₂⁻) by the NADH dehydrogenase in bovine heart mitochondria. Submitochondrial particles, when reduced by NADH and rotenone or through 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 NADH and antimycin but inhibited by rotenone, antimycin, and cyanide. Rotenone has a biphasic effect, increasing O₂⁻ production at the NADH dehydrogenase site and inhibiting it at the ubiquinone–cytochrome b site. The generation of O₂⁻ by the NADH dehydrogenase is about 50% of the rate at the ubiquinone–cytochrome b site. The pH dependence of O₂⁻ production shows a second maximum at pH 8.6–8.8, close to the optimal pH for the ubiquinone–cytochrome b site. The study also describes an O₂⁻-dependent autocatalytic process involving adrenaline, NADH, and submitochondrial particles. The results confirm the presence of two sites for O₂⁻ production in the mitochondrial respiratory chain and highlight the role of the NADH dehydrogenase in O₂⁻ generation.The study by Turrens and Boveris investigates the generation of superoxide anion (O₂⁻) by the NADH dehydrogenase in bovine heart mitochondria. Submitochondrial particles, when reduced by NADH and rotenone or through 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 NADH and antimycin but inhibited by rotenone, antimycin, and cyanide. Rotenone has a biphasic effect, increasing O₂⁻ production at the NADH dehydrogenase site and inhibiting it at the ubiquinone–cytochrome b site. The generation of O₂⁻ by the NADH dehydrogenase is about 50% of the rate at the ubiquinone–cytochrome b site. The pH dependence of O₂⁻ production shows a second maximum at pH 8.6–8.8, close to the optimal pH for the ubiquinone–cytochrome b site. The study also describes an O₂⁻-dependent autocatalytic process involving adrenaline, NADH, and submitochondrial particles. The results confirm the presence of two sites for O₂⁻ production in the mitochondrial respiratory chain and highlight the role of the NADH dehydrogenase in O₂⁻ generation.