Because we previously reported that T₃ and 3,5-diiodo-l- thyronine (3,5-T₂) both increase resting metabolic rate (RMR), 3,5-T₂ could be another thyroidal regulator of energy metabolism. This effect of 3,5-T₂ is evident in rats made hypothyroid by propylthiouracil and iopanoic acid, not in normal euthyroid (N) rats. Possibly, under euthyroid conditions, active 3,5-T₂ may need to be formed intracellularly from a precursor such as T₃. We tested this hypothesis by giving a single injection of T₃ to N rats and comparing the time course of the variations in RMR with those of the changes in the serum and hepatic levels of 3,5-T₂. Acute injection had an evident effect on RMR, 25 h earlier, in N rats than in rats made hypothyroid by propylthiouracil and iopanoic acid, maximal values (+40%) being reached in the former at 24–26 h. In N rats, the simultaneous injection of actinomycin D with the T₃ inhibited the late part of the effect (after 24 h) more strongly than the early part (14–24 h). In serum and liver, 3,5-T₂ levels were increased significantly at 12–24 h after T₃ injection into N rats, a time at which RMR was rising rapidly to peak. These results seem to indicate that when T₃ is injected into N animals, not all the effects on RMR are attributable to T₃ itself, the early effect presumably being largely because of its in vivo deiodination to 3,5-T₂. Because the effects of T₃ and 3,5-T₂ are additive, in N rats, the two iodothyronines probably cooperate in vivo to determine the total metabolic rate.