The local conversion of thyroxine (T4), which is an important source of intracellular 3,5,3'-triiodothyronine (T3) in several rat tissues, has been subject of recent investigations. In the present study the regulation of this phenomenon in vivo was investigated in various peripheral tissues of the rat. Intact euthyroid and radiothyroidectomized (Tx) rats received a continuous intravenous infusion of [125I]T4 and [131I]T3 until isotope equilibrium was attained. In addition to the labeled iodothyronines, Tx rats received a continuous intravenous infusion of 0.2 or 1.0 microgram carrier T4/100 g body wt per d, to create hypothyroid or slightly hypothyroid conditions, respectively. After the animals were bled and perfused the contribution of T3 derived from local conversion of T4 to T3 [Lc T3(T4)] to the total T3 in homogenates from several tissues and subcellular fractions from the liver, kidney, and anterior pituitary gland could be calculated. In all experiments T3 in muscle was derived exclusively from the plasma. In the cerebral cortex and cerebellum, however, most of the intracellular T3 was derived from the intracellular conversion of T4 to T3. It is demonstrated that for hypothyroid rats an increased relative contribution of Lc T3(T4) reduced the loss of total T3 in the brain. This phenomenon was also encountered for the anterior pituitary gland, although in this tissue the proportion of the total tissue T3, contributed by locally produced T3 was considerably lower than the values found for the cerebral cortex and cerebellum in all experiments. The present findings, regarding the source and quantity of pituitary nuclear T3 strongly suggest that both plasma T3 and T4 (through its local conversion into T3) play a role in the regulation of thyrotropin secretion. The contribution of Lc T3(T4) to the total pituitary nuclear T3 was of minor importance in euthyroid rats (approximately 20%), compared with that found for both groups in T4-supplemented athyreotic rats (approximately 40%). The total T3 concentration in the liver decreased from euthyroid to hypothyroid rats and was associated with a decrease in the tissue/plasma T3 concentration gradient. A minor proportion of hepatic T3 was contributed by Lc T3(T4), which in fact decreased significantly from the euthyroid to the hypothyroid state. In contrast to other subcellular fractions from the liver, no Lc T3(T4) could be demonstrated in the nuclear fraction. It is suggested that the liver plays an important role with respect to regulation of the circulating T3 concentration. In the kidney, a very small proportion of the total T3 was derived from locally produced T3 in all experiments (4-7%). As found in the liver, all nuclear T3 appeared to be derived from the plasma. In contrast to the liver, subcellular T3 pools in the kidney seemed to be exchangeable.