The mitochondrial cytochrome bc(1) complex (ubiquinol/cytochrome c oxidoreductase) is generally thought to generate superoxide anion that participates in cell signaling and contributes to cellular damage in aging and degenerative disease. However, the isolated, detergent-solubilized bc(1) complex does not generate measurable amounts of superoxide except when inhibited by antimycin. In addition, indirect measurements of superoxide production by cells and isolated mitochondria have not clearly resolved the contribution of the bc(1) complex to the generation of superoxide by mitochondria in vivo, nor did they establish the effect, if any, of membrane potential on superoxide formation by this enzyme complex. In this study we show that the yeast cytochrome bc(1) complex does generate significant amounts of superoxide when reconstituted into phospholipid vesicles. The rate of superoxide generation by the reconstituted bc(1) complex increased exponentially with increased magnitude of the membrane potential, a finding that is compatible with the suggestion that membrane potential inhibits electron transfer from the cytochrome b(L) to b(H) hemes, thereby promoting the formation of a ubisemiquinone radical that interacts with oxygen to generate superoxide. When the membrane potential was further increased, by the addition of nigericin or by the imposition of a diffusion potential, the rate of generation of superoxide was further accelerated and approached the rate obtained with antimycin. These findings suggest that the bc(1) complex may contribute significantly to superoxide generation by mitochondria in vivo, and that the rate of superoxide generation can be controlled by modulation of the mitochondrial membrane potential.