Uncoupling protein (UCP1) is a transmembrane proton transporter present in the mitochondria of brown adipose tissue (BAT), a specialized tissue which functions in temperature homeostasis and energy balance (Nicholls, D. G., and Locke, R. M. (1984) Physiol. Rev. 64, 2-40; Lowell, D. D., and Flier, J. S. (1997) Annu. Rev. Med.). UCP1 mediates the thermogenesis that is characteristic of BAT by uncoupling mitochondrial oxidation of substrates from ATP synthesis. Recently, two proteins related to UCP1 have been identified and designated UCP2 (Fleury, C., et al. (1997) Nature Genetics 15, 269-272) or UCP homolog (UCPH) (Gimeno, R. E., et al. (1997) Diabetes 46, 900-906) and UCP3 (Boss, O., et al. (1997) FEBS Lett. 408, 39-42; Vidal-Puig, A., et al. (1997) Biochem. Biophys. Res. Commun. 235, 79-82). We investigated the regulation in rats of UCP3, which is expressed primarily in skeletal muscle and BAT. Expression of rat UCP3 mRNA in BAT was upregulated by in vivo treatment with triiodothyronine (T3) and by exposure to cold, suggesting that UCP3 is active in thermogenesis and energy expenditure. In skeletal muscle, UCP3 mRNA was also upregulated by T3 but, surprisingly, not by cold exposure. A hypothesis is proposed to account for this differential regulation.