Growing evidence shows that, among triiodothyronine derivatives, 3,5 diiodo-L-thyronine (T(2)) plays an important role in energy metabolism and fat storage. In the present study, short-term effects of T(2) administration to hypothyroid rats on fatty acid oxidation rate and bioenergetic parameters were investigated. Within 1 h following T(2) injection, state 3 and state 4 respiration rates, which were reduced in hypothyroid mitochondria, were noticeably increased particularly in succinate- with respect to glutamate/malate-energized mitochondria. Maximal respiratory activity, observed when glutamate/malate/succinate were simultaneously present in the respiratory medium, was significantly stimulated by T(2) treatment. A T(2)-induced increase in respiratory rates was also observed when palmitoyl-CoA or L-palmitoylcarnitine were used as substrates. No significant change in respiratory control index and ADP/O ratio was observed. The activities of the mitochondrial respiratory chain complexes, especially Complex II, were increased in T(2)-treated rats. In the latter, Complex V activities, assayed in both ATP synthesis and hydrolysis direction, were enhanced. The rate of fatty acid oxidation, followed by conversion of [(14)C]palmitate to CO(2) and ketone bodies, was higher in hepatocytes isolated from T(2)-treated rats. This increase occurs in parallel with the raise in the activity of carnitine palmitoyltransferase-I, the rate limiting enzyme of fatty acid β-oxidation, assayed in situ in digitonin-permeabilized hepatocytes. Overall, these results indicate that T(2) rapidly increases the ability of mitochondria to import and oxidize fatty acids. An emerging idea in the literature is the ability of T(2) to reduce adiposity and dyslipidemia and to prevent the development in liver steatosis. The results of the present study, showing a rapid T(2)-induced increase in the ability of mitochondria to import and oxidize fatty acids, may contribute to understand the biochemical mechanisms of T(2)-metabolic effects.