The objective of the present study was to test in vivo the metabolic effects of 3,5-di-iodothyronine (3,5-T2) in unanesthetized and unrestrained male Sprague–Dawley rats. Amino acid and lipid metabolisms were investigated by breath tests using as tracers the 13C-carboxyl-labeled molecules of leucine, α-ketoisocaproic acid (KIC) and octanoic acid, in four different groups of rats: hypothyroid animals (receiving propylthiouracil (PTU) and iopanoic acid), hypothyroid animals treated with either a daily i.p. injection of 3,5-T2 (25 μg/100 g body weight), or triiodothyronine (T3) (1 μg/100 g body weight), and control euthyroid animals receiving equivalent volumes of the vehicle solutions. Energy expenditure was measured by continuous monitoring of O2 consumption and CO2 production in these different groups. Daily energy expenditure was decreased by 30% in PTU-treated rats. The chronic treatments with 3,5-T2 and T3 restored daily energy expenditure to the control level. 13CO2 recovered in breath following the i.v. injection of octanoic acid-[1-13C] was decreased in hypothyroid animals compared with control animals (P<0·05) and restored to control values by T3 and 3,5-T2 treatments. The 13CO2 recovered in breath after i.v. injection of leucine-[1-13C]was increased in PTU-treated compared with control animals (P<0·05). Chronic treatment with either 3,5-T2 or T3 restored 13CO2 to control values. Excretion of 13CO2 recovered in breath following the i.v. injection of KIC-[1-13C] was increased in PTU-treated compared with control animals. Chronic treatments with either 3,5-T2 or T3 did not restore KIC decarboxylation. These results suggest that 3,5-T2 exerts metabolic effects on energy expendi ture, on both lipid β-oxidation and leucine metabolism in hypothyroid rats. We conclude that 3,5-T2 is a metabolically active iodothyronine.