An in vitro model has been employed to study the nature of the process of peripheral monodeiodination of thyroxine to triiodothyronine (T3) and the factors capable of modulating it. Thyroxine (T4, 5 μg/ml, 6 x 10-6M) was incubated in 0.15M phosphate buffer (pH 7.35) with various rat tissue homogenates (approximately 0.13 g-equivalent) for 2 h at 37 C, and the T3 generated during incubation was measured by a specific immunoassay of an ethanol extract of the homogenate. The liver and kidney homogenates produced more T3 than various other tissues; the kidney was more active than the liver. The T4 to T3 converting activity in the liver homogenates was influenced by the concentration of the homogenate, duration of incubation, substrate (T4) concentration, pH of incubation and temperature of incubation. It was unaffected by large concentrations of several agents including methimazole, hydrocortisone, sodium iodide, mono- or diiodotyrosine, 3,5-diidothyronine, thyronine and methylated and halogenated analogues of 3,3',5'-triidothyronine (reverse T3, rT3). However, various other agents including several thyroid analogues and propylthiouracil (PTU) inhibited T4 to T3 conversion in a dose-dependent manner. Inhibitory thyroid analogues, in order of their potency, were rT3, 3',5'-diiodothyronine, tetraiodothyroacetic acid, 3,3'-diiodothyronine and 3-monoiodothyronine; on a molar basis, the relative potency of these agents was approximately 100:100:5:1:1. PTU was about 3% as potent as rT3 on a weight basis and only 1% as potent as rT3 on a molar basis. Analyses of the data by Lineweaver-Burk plot suggested that rT3 is a competitive inhibitor and PTU, an uncompetitive inhibitor of conversion of T4 to T3. The various data suggest that: a) monodeiodination of T4 to T3 is enzymic in nature, and b) rT3 is a very potent inhibitor of conversion of T4 to T3.