Uncoupling protein-3 (UCP3) is a mitochondrial carrier protein of as yet undefined physiological function. To elucidate characteristics of its function, we studied the effects of fasting on resting metabolic rate, respiratory quotient, muscle Ucp3 expression, and mitochondrial proton leak in wild-type and Ucp3(-/-) mice. Also analyzed were the fatty acid compositions of skeletal muscle mitochondria in fed and fasted Ucp3(-/-) and wild-type mice. In wild-type mice, fasting caused significant increases in Ucp3 (4-fold) and Ucp2 (2-fold) mRNA but did not significantly affect mitochondrial proton leak. State 4 oxygen consumption was not affected by fasting in either of the two groups. However, protonmotive force was consistently higher in mitochondria of Ucp3(-/-) animals (P = 0.03), and fasting further augmented protonmotive force in Ucp3(-/-) mice; there was no effect in wild-type mitochondria. Resting metabolic rates decreased with fasting in both groups. Ucp3(-/-) mice had higher respiratory quotients than wild-type mice in fed resting states, indicating impaired fatty acid oxidation. Altogether, results show that the fasting-induced increases in Ucp2 and Ucp3 do not correlate with increased mitochondrial proton leak but support a role for UCP3 in fatty acid metabolism.