The function of uncoupling protein 3 is still not established. Mitochondrial uncoupling, control of ROS production, protection against lipotoxicity and protection against oxidative stress are functions classically discussed. To establish a role for uncoupling protein 3 in these functions, we have here used uncoupling protein 3 (-/-) mice, backcrossed for 10 generations on a C57Bl/6 background. In isolated skeletal muscle mitochondria, we examined uncoupled respiration, both unstimulated and in the presence of fatty acids. We did not observe any difference between mitochondria from wildtype and uncoupling protein 3(-/-) mice. We measured H(2)O(2) production rate and respiration rate under reactive oxygen species-generating conditions (succinate without rotenone) but found no effect of uncoupling protein 3. We tested two models of acute lipotoxicity-fatty acid-induced oxidative inhibition and fatty acid-induced swelling-but did not observe any protective effect of uncoupling protein 3. We examined oxidative stress by quantifying 4-hydroxynonenal protein adducts and protein carbonyls in the mitochondria-but did not observe any protective effect of uncoupling protein 3. We conclude that under the experimental conditions tested here, we find no evidence for the function of uncoupling protein 3 being basal or induced uncoupling, regulation of ROS production, protection against acute lipotoxicity or protection against oxidative damage.