Metabolically active brown adipose tissue (BAT) plays an exclusive role in maintaining body energy homeostasis by adaptive thermogenesis through the UCP1-mediated mitochondrial uncoupling. 3,5-diiodo-l-thyronine (3,5-T2), a thyroid hormone derivative, enhances rat metabolic rate and prevents diet induced obesity. The tissues underlying the metabolic effect of T2 are only partially identified, and BAT, in view of its physiological role, could be a target for T2. We evaluated the ability of T2 to activate BAT and we used hypothyroid and T2 treated- hypothyroid rats maintained at thermoneutrality. T2 administration into hypothyroid rats enhances their -heat production - BAT mass/rat mass -BAT mitochondrial content. Immunohistochemical analyses indicate that T2 enhances BAT vascularization and sympathetic innervation, as well as the content of cytochrome oxidase subunits IV (a marker of mitochondrial content) and UCP1. At the functional level, T2 enhances respiration rate in isolated mitochondria. This effect seems to be mediated by UCP1 as it was abolished by the "in vitro" addition of GDP (an UCP1 inhibitor) to mitochondria and restored by the presence of arachidonate (which removes the inhibition exerted by GDP on UCP1). These data clearly indicate that BAT is a target for T2 and underlies some of its metabolic effects.