To investigate the role of type II 5′-deiodinase (5′D-II) in the expression of mitochondrial uncoupling protein (UCP) in brown adipose tissue (BAT), we injected intact male rats with reverse (r) 3,5,3′-triiodothyronine (T₃; 100 μg ⋅ 100 g body wt⁻¹ ⋅ day⁻¹), an inhibitor of 5′D-II, for 2–5 days. UCP decreased by ∼20% in rats kept at 28°C and failed to increase during cold exposure (4°C). Next, thyroxine treatment (1–10 μg ⋅ 100 g body wt⁻¹ ⋅ day⁻¹) increased nuclear T₃ in rats kept at 28 or 4°C. In these rats, nuclear T₃ correlated positively with UCP. In addition, T₃ (1–50 μg ⋅ 100 g body wt⁻¹ ⋅ day⁻¹) given to intact rats (5–15 days; 28°C) induced an approximately twofold increase in UCP. In these T₃-treated animals, the interscapular BAT thermal response to norepinephrine infusion also correlated positively with T₃ dose and UCP content. Treatment with propranolol or reserpine failed to block the T₃ induction of UCP (∼1.8- and ∼2.3-fold). The results emphasize the importance of local 5′D-II and reveal an independent role of T₃ in the expression of UCP.