Mitochondrial uncoupling protein (UCP) 1 has been thought to be a key molecule for thermogenesis during cold exposure and spontaneous hyperphagia. However, UCP1-knockout (KO) mice were reported to be cold-intorelant, but unexpectedly not to get obese even after hyperphagia, implying that UCP1 may not involved in the regulation of adipocity. Treatment of obese animals with 3-adrenergic agonists is known to increase lipid mobilization, induce UCP1, and finally reduce body fat content. To obtain direct evidence for the role of UCP1 in the anti-obesity effect of 3-adrenergic stimulation, UCP1-KO and wild-type (WT) mice were fed on cafeteria-diets for 8 weeks, and then given a 3-adrenergic agonist CL316,243 (CL) for 2 weeks. Single injection of CL increased whole body oxygen consumption and brown fat temperature in WT mice, but not in KO mice, while it elicited almost the same plasma free fatty acid response in WT and KO mice. WT and KO mice increased similarly their body and white fat pad weights on cafeteria-diets. Daily treatment with CL reduced white fat pad weight and the size of adipocytes in WT mice, but not in KO mice. Compared to WT mice, KO mice expressed increased levels of UCP2 in brown fat but decreased levels in white fat, and comparable levels of UCP3 in skeletal muscle. It was concluded that the anti-obesity effect of 3-adrenergic stimulation is largely attributable to UCP1, but little to UCP2 and UCP3, and thereby to UCP1-dependent degradation of fatty acids released from white adipose tissue.