The relationship between protonmotive force and superoxide production by mitochondria is poorly understood. To address this issue, the rate of superoxide production from complex I of rat skeletal muscle mitochondria incubated under a variety of conditions was assessed. By far, the largest rate of superoxide production was from mitochondria respiring on succinate; this rate was almost abolished by rotenone or piericidin, indicating that superoxide production from complex I is large under conditions of reverse electron transport. The high rate of superoxide production by complex I could also be abolished by uncoupler, confirming that superoxide production is sensitive to protonmotive force. It was inhibited by nigericin, suggesting that it is more dependent on the pH gradient across the mitochondrial inner membrane than on the membrane potential. These effects were examined in detail, leading to the conclusions that the effect of protonmotive force was mostly direct, and not indirect through changes in the redox state of the ubiquinone pool, and that the production of superoxide by complex I during reverse electron transport was at least 3-fold more sensitive to the pH gradient than to the membrane potential.