A role for uncoupling protein (UCP) homologues in mediating the proton leak in mammalian mitochondria is controversial. We subjected insulinoma (INS-1) cells to adenoviral expression of UCP2 or UCP1 and assessed the proton leak as the kinetic relationship between oxygen use and the inner mitochondrial membrane potential. Cells were infected with different amounts of rat UCP2, and, in other experiments, with either UCP2 or UCP1. The relative molar expression of these subtypes was quantified through comparison with histidine-tagged UCP1 or UCP2 proteins engineered by expression in Escherichia coli. Adenoviral infection with UCP2, compared with beta-galactosidase, resulted in a dose-dependent shift in kinetics indicating increased H(+) flux at any given membrane potential. UCP1 also enhanced H(+) flux, but, on a relative molar basis, the overexpression of the endogenous protein, UCP2, was more potent than UCP1. These results were not due to nonspecific overexpression of mitochondrial protein since UCP1 activity was inhibited by GDP and because overexpression of another membrane carrier protein, the oxoglutarate malate carrier had no effect. UCP2-mediated H(+) conduction was not GDP sensitive. These data suggest that the UCP homologue, UCP2, mediates the proton leak in mitochondria of a mammalian cell wherein UCP2 is the native subtype.