Addition of coenzyme Q(10) (CoQ) at low concentration (29 nmol/mg of protein) to kidney but not liver mitochondria resulted in an increase in proton conductance. This uncoupling activity required fatty acid and was completely inhibited by GDP. CoQ activated when it was likely to be reduced but not when it was likely to become oxidized. However, the redox state of endogenous CoQ did not affect mitochondrial proton conductance. Stimulation by CoQ was not inhibited by cyclosporin A, carboxyatractylate, bongkrekate and catalase but could be reversed by superoxide dismutase. We conclude that CoQ acted in mitochondria through production of superoxide, which mediated uncoupling, probably by acting through uncoupling protein 2.