This work was undertaken to study the action exerted by thyroid hormones on mitochondria. By day 6 after thyroidectomy, the respective activities of two inner-membrane enzymes--succinate and beta-hydroxybutyrate cytochrome c reductases--had already dropped by 32 and 50%, whereas, in the outer membrane, the activity of rotenone-insensitive NADH-cytochrome c reductase did not change significantly. The decrease in the activity of the inner-membrane enzymes closely followed the disappearance of T3 and T4 from serum. 10 h after administration of 25 micrograms/100 g T3 to thyroidectomized rats, the activity of succinate and beta-hydroxybutyrate cytochrome c reductases and the oxygen consumption rate with succinate or beta-hydroxybutyrate were significantly increased, while, in the outer membrane, the activity of monoamine oxidase and rotenone-insensitive NADH-cytochrome c reductase remained unchanged. In the thyroidectomized rat, L-[3H]leucine incorporation in vivo is diminished in all the liver mitochondrial proteins, and especially in two constituents of MW 19 000 and 28 000. The radioactivity of these two components is also decreased in the normal rat treated with chloramphenicol, a specific inhibitor of mitochondrial protein synthesis. L-[14C]leucine incorporation in isolated liver mitochondria was significantly increased in the thyroidectomized rat, 10 h after T3 treatment. Thus, thyroid hormones have an early and preferential action on the mitochondrial protein synthesizing system and on the inner-membrane enzyme activities.