We sought a correlation between rat skeletal muscle triiodothyronine (T3)-mediated regulation of uncoupling protein-3 (UCP3) expression and mitochondrial activity. UCP3 mRNA expression increased strongly during the hypothyroid-hyperthyroid transition. The rank order of mitochondrial State 3 and State 4 respiration rates was hypothyroid < euthyroid < hyperthyroid. The State 4 increase may have been due to the increased UCP3 expression, as the proton leak kinetic was stimulated in the hypothyroid-hyperthyroid transition and a good correlation exists between the State 4 and UCP3 mRNA level. As a significant proportion of an organism's resting oxygen consumption is dedicated to opposing the proton leak, skeletal muscle mitochondrial UCP3 may mediate part of T3's effect on energy metabolism.