Uncoupling protein (UCP) is essential to brown adipose tissue (BAT) thermogenesis and, hence, to cold adaptation and energy balance. The sympathetic nervous system, via norepinephrine and cAMP, and thyroid hormone seem to be the major regulators of UCP expression. T3 potentiates the effect of norepinephrine and is essential for the adaptive response of this protein to cold. The goal of the present studies was to investigate whether T3 directly stimulates the transcription of the rat UCP gene, as suggested by in vivo results, and if so, to identify and characterize the sequences involved. We examined the gene sequence between 114 and -3623 by transient transfection analysis in JEG-3 and HIB-1B cells, a BAT-derived cell line. This 3.7-kilobase UCP insert makes the reporter gene responsive to cAMP (4-fold), T3 (4-fold), or both combined (12-fold). We identified an 82-basepair (bp) restriction fragment between -2317 and -2399, which we called thyroid hormone response sequence (THRS), that conferred T3 responsiveness to the UCP minimal promoter (4- to 12-fold) as well as to the thymidine kinase promoter (3- to 6-fold). T3 receptor bound to THRS in vitro, retarding its migration in electrophoretic mobility shift assays. Footprinting of THRS revealed two potential thyroid hormone response elements (TRE) separated by 27 bp: upTRE, -2391/-2376, 5'ACCCCTACTGAGGCAA; and dnTRE, -2348/-2334, 5'AGGGCAGCAAGGTCA. The mutation of these putative TREs caused loss of both T3 receptor binding and transactivation by T3. The analysis of the mutants also demonstrated that both TREs contribute in similar proportion to the T3 responsiveness of the UCP gene and that dnTRE is necessary for the potentiation of the cAMP effect by T3. Both TREs are located within a previously identified 212-bp enhancer element, flanked by sequences considered essential for BAT expression and norepinephrine responsiveness. Although they do not mediate thyroid hormone responsiveness, the sequences flanking THRS increase basal reporter expression and enhance the responses to T3. In conclusion, our results indicate that T3 can stimulate the transcription of the UCP gene and amplify the effect of cAMP acting directly on the gene. The presence of two functional TREs in a location critical to the control of the gene supports the importance of thyroid hormone for its expression and suggests the potential for interactions at the gene level that may explain the complexity of UCP regulation in vivo.