The thyroid hormone, triiodothyronine, has been shown to be bound by the intranuclear chromatin protein associated with active DNA, where it is believed to stimulate transcription. Evidence exists that the thyroid hormones have direct action not only on nuclei, but also on mitochondria. Therefore, specific proteins that bind thyroid hormones in the mitochondria should be demonstrable.Mitochondria were isolated from homogenized rat livers by sedimentation through 0.25 M sucrose solution, followed by washing four times to free them of microsomes The many recent reports from the laboratories of Oppenheimer (8-15), , and de Groot (5-7) provide evidence of specific nuclear receptors for the thyroid hormones, thyroxine (T4) and triiodothyronine (TS), and suggest that the cell nucleus is a significant locus of hormone action. Indeed, as early as 1966, Siegel and Tobias demonstrated the nuclear localization of thyroid hormone by radioautography of tritium-labeled hormone added to cell cultures of human renal epithelial cells, grown in monolayer (31, 32), an observation which forecast the later developments. These authors showed a diminution of the number of tritium grains over the nucleus if actinomycin D had been added to the cell cultures, but an increase after addition of puromycin, a finding compatible with thyroid hormone action at the transcriptional level in the nucleus.Prior to and throughout the same period of time, however, Tapley and his associates (33-42) have been accumulating evidence that suggests direct thyroid hormone action upon the mitochondria of responsive cells. Binding of thyroid hormones by rnitochondrial membranes was confirmed by Tata, Ernster, and Suranyi, who reported microsomal and nuclear binding as well (43). Perhaps the most striking observation has been the demonstration of increased mitochondrial protein synthesis in as short an interval as 3 min after the addition of T4 or T3 to isolated rat liver mitochondria in vitro (35).Recently we have been studying the binding of T4 and T3 to cell proteins (23,24). The present communication concerns the finding of a protein fraction of the mitochondrial membrane which binds T4 and T3 in the physiological range, and is presumed related to hormonal effect upon energy metabolism.After isolation of mitochondria from rat liver and kidney, the binding of labeled hormones by intact mitochondria was studied, followed by examination of the mitochondrial matrix and membranes, and subsequently protein fractions extracted from the mitochondrial membranes.