Triiodothyronine regulates energy metabolism and thermogenesis. Among triiodothyronine derivatives, 3,5-diiodo-l-thyronine (T(2)) has been shown to exert marked effects on energy metabolism by acting mainly at the mitochondrial level. Here we investigated the capacity of T(2) to affect both skeletal muscle mitochondrial substrate oxidation and thermogenesis within 1 h after its injection into hypothyroid rats. Administration of T(2) induced an increase in mitochondrial oxidation when palmitoyl-CoA (+104%), palmitoylcarnitine (+80%), or succinate (+30%) was used as substrate, but it had no effect when pyruvate was used. T(2) was able to 1) activate the AMPK-ACC-malonyl-CoA metabolic signaling pathway known to direct lipid partitioning toward oxidation and 2) increase the importing of fatty acids into the mitochondrion. These results suggest that T(2) stimulates mitochondrial fatty acid oxidation by activating several metabolic pathways, such as the fatty acid import/beta-oxidation cycle/FADH(2)-linked respiratory pathways, where fatty acids are imported. T(2) also enhanced skeletal muscle mitochondrial thermogenesis by activating pathways involved in the dissipation of the proton-motive force not associated with ATP synthesis ("proton leak"), the effect being dependent on the presence of free fatty acids inside mitochondria. We conclude that skeletal muscle is a target for T(2), and we propose that, by activating processes able to enhance mitochondrial fatty acid oxidation and thermogenesis, T(2) could play a role in protecting skeletal muscle against excessive intramyocellular lipid storage, possibly allowing it to avoid functional disorders.