We investigated the role of uncoupling protein 3 (UCP3) during fasting and examined the effect of triiodothyronine (T3) administration in such a condition. The possible involvement of mitochondrial thioesterase (MTE I) and the role of putative cofactors, such as coenzyme Q (CoQ), was also examined. Here, we report that fasting induced a more than twofold elevation in the expression and activity of MTE I, and an increase in UCP3 expression, without any associated uncoupling activity. Administration of T3 to fasting rats further up-regulated UCP3 as well as MTE I expression, markedly enhanced MTE I enzyme activity and prevented the impairment of the uncoupling activity of UCP3 normally seen during fasting. Indeed, T3-treatment induced an UCP3-dependent decrease in mitochondrial membrane potential, which was abolished by the addition of either GDP or superoxide dismutase (SOD). T3 administration also prevented the marked decrease of CoQ levels observed in fasting rats and this provides evidence that also, in vivo, CoQ represents an essential cofactor for the UCP3-mediated uncoupling. The data also show that MTE I and UCP3 are likely involved in the same biochemical mechanism and that UCP3 postulated functions, such as lipid handling and uncoupling, are not mutually exclusive but may coexist in vivo.