Isolated hepatocytes from euthyroid or hypothyroid (thyroidectomized) rats showed a 20 to 30% stimulation of respiratory activity when treated with triiodothyronine (T₃). Maximal stimulation was obtained in the presence of the uncoupler carbonylcyanide m-chlorophenyl-hydrazone (CCCP) suggesting a direct effect on the respiratory chain. Physiological concentrations of T₃ (10^-11-10^-9) induced maximal stimulation after a short incubation (75% of maximum in 3 min) and the effect persisted for over 1 h of incubation. Mitochondria, rapidly isolated from T₃-treated hepatocytes, retained stimulated respiratory activity in both coupled and uncoupled states. These results are consistent with the hypothesis that the mitochondrion is a primary target for thyroid hormones, and the present system appears to be suitable for the systematic investigation of these effects.