Serum triiodothyronine (T(3)) kinetics in man have been difficult to define presumably due to the interference of iodoproteins generated during the peripheral metabolism of T(3). The use, in the present study, of an anion-column chromatographic method for separation of serum T(3) as well as thyroxine (T(4)) from these iodoproteins has overcome this technical handicap. Simultaneous measurement of serum (125)I-T(3) and (131)I-T(4) kinetics were performed in 31 subjects from the clinical categories of euthyroid, primary hypothyroid, thyrotoxic and posttreatment hypothyroid Graves' disease, factitial thyrotoxic, and idiopathically high and low thyroxinebinding globulin states. The normal mean T(3) fractional turnover rate (kT(3)) was 0.68 (half-life = 1.0 days), increased in toxic Graves' disease patients to 1.10 (half-life = 0.63 days), and decreased in primary hypothyroid patients to 0.50 (half-life = 1.38 days). The mean T(3) equilibration time averaged 22 hr except in hypothyroid and high thyroxine-binding globulin (TBG) patients where the equilibration period was delayed by 10 hr. The mean T(3) distribution space in normal subjects was 38.4 liters. This was reduced in subjects with high TBG levels (26 liters) and increased in patients with low TBG and in all hyperthyroid states (53-55 liters). The normal serum T(3) concentration was estimated by radioimmunoassay to be 0.106 mug/100 ml. Combined with the mean T(3) clearance value of 26.1 liters/day, the calculated T(3) production rate was 27.6 mug/day. The mean T(3) production rate increased to 201 mug/day in thyrotoxic Graves' disease patients and was reduced to 7.6 mug/day in primary hypothyroid subjects. T(3) production rate was normal in subjects with altered TBG states. The ratio of T(3) to T(4) production rate in normal subjects was 0.31 and was unchanged in patients with altered TBG values. This ratio was increased in all Graves' disease patients with the highest value being 0.81 in the posttreatment hypothyroid Graves' disease group. This apparent preferential production of T(3) may have been responsible for the retention of rapid turnover kinetics for T(3) and T(4) observed in treated Graves' disease patients. The finding that factitial thyrotoxic patients also displayed similar rapid T(3) and T(4) turnover kinetics indicates that these alterations are not a unique feature of Graves' disease per se. When comparing the peripheral turnover values for T(3) and T(4) in man, it is apparent that alterations in metabolic status and serum TBG concentration influence both hormones in a parallel manner; however, changes in metabolic status seem to have a greater influence on T(3) kinetics while alterations in TBG concentrations have a greater effect on T(4). These observations probably relate to the differences in TBG binding affinity and peripheral tissue distribution of these two hormones.