Triiodothyronine regulates energy metabolism and thermogenesis. Among triiodothyronine derivatives, 3,5-diiodo-L-thyronine (T₂) has been shown to exert marked effects on energy metabolism by acting mainly at the mitochondrial level. Here we investigated the capacity of T₂ to affect both skeletal muscle mitochondrial substrate oxidation and thermogenesis within 1 hour after its injection into hypothyroid rats. Administration of T₂ induced an increase in mitochondrial oxidation when palmitoyl-CoA (+104%), palmitoyl-carnitine (+80%), or succinate (+30%) was used as substrate, but it had no effect when pyruvate was used. T₂ was able a) to activate the AMPK-ACC-Malonyl CoA metabolic signalling pathway known to direct lipid partitioning towards oxidation and (b) to increase the importing of fatty acids into the mitochondrion. These results suggest that T₂ stimulates mitochondrial fatty acid oxidation by activating additional metabolic pathways: import of fatty acids into the mitochondrion -- beta oxidation cycle -- FADH2-linked respiratory pathways. T₂ 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 T₂, and we propose that, by activating processes able to enhance mitochondrial fatty acid oxidation and thermogenesis , T₂ could play a role in protecting skeletal muscle against excessive intramyocellular lipid storage, possibly allowing it to avoid functional disorders.