Many studies suggest that one enzyme is involved in the phenolic ring deiodination of iodothyronines in rat liver and kidney and another one in the tyrosyl ring deiodination. This study describes some characteristic of the phenolic ring (5'-) deiodination of rT3 and 3',5'-T2 by rat liver microsomes. At pH 7.2 the Km values of the 5'-deiodination of rT3 and 3',5'-T2 were 0.103 and 0.77 microM, respectively. 3',5'-T2 and rT3 inhibited the respective 5'-deiodination reactions competitively, the Ki values being 1.05 and 0.134 microM, respectively. Several radiographic contrast agents markedly inhibit the 5'-monodeiodination or rT3 and 3',5'-T2, the type of inhibition being competitive. Of these compounds iopanoic acid, ipodic acid and iophenoxic acid are the most potent inhibitors with Ki values of approximately 2 microM for both reactions. The non-iodine containing compound 8-anilino-1-naphthalene sulphonic acid (ANS) appeared to be a very strong competitive inhibitor of both 5'-deiodinations (Ki 4.3-4.7 microM), whereas salicylic acid, which as ANS inhibits the binding of iodothyronines to T4-binding globulin, inhibited these reactions to a much lesser extent (Ki 300-500 microM). On the other hand, diiodosalicylic acid was a very strong inhibitor. The beta-adrenergic blocker D,L-propranolol was a weak noncompetitive inhibitor of both 5'-deiodinations (Ki 0.4-0.7 mM). These reactions were also inhibited by various 2,6-diiodophenol derivatives, triiodophenol being the strongest and diiodotyrosine the weakest inhibitor tested. Comparing the Ki values of various inhibitors for the 5'-deiodination of rT3 and 3',5'-T2, a positive correlation between these values was found (r = 0.97). It was concluded that rT3 (to 3,3'-T2) and 3'-5'-T2 (to 3'-T1) monodeiodinating activities are very similar to each other and that there may just be one monodeiodinase catalyzing the 5'-deiodination of iodothyronines in rat liver.