The enzymatic conversion of thyroxine (T4) to 3,5,3-triiodothyronine (T3) by iodothyronine 5'-deiodinase(s) is an obligate step in the physiologic action of thyroid hormones in most extrathyroidal tissues. In the rat liver and kidney, 5'-deiodinase processes having either high Km (micromolar range) or low Km (nanomolar range) values for thyroid hormone substrates have been described. The number of enzymes mediating these reactions, however, remains uncertain and controversial. To examine this question we have compared the 5'-deiodinase activity expressed in membrane preparations of Xenopus laevis oocytes after the injection of either rat liver poly(A)+ RNA or in vitro prepared RNA transcribed using the G21 full-length type I 5'-deiodinase cDNA. In oocytes injected with rat liver poly(A)+ RNA, high Km (i.e. type I) activity was observed when 20 mM dithiothreitol was used as the thiol cofactor, whereas Km values in the nanomolar range were noted with 0.5 mM dithiothreitol, glutathione, or a reconstituted thioredoxin cofactor system. This complex pattern of 5'-deiodinase activity, which mimics that found in homogenates and subcellular fractions of rat liver and kidney, was reproduced exactly in oocytes by the microinjection of G21-derived RNA transcripts. Furthermore, hybrid arrest of translation in oocytes using a partial type I 5'-deiodinase cDNA completely inhibited the expression of both high and low Km activity after the injection of rat liver poly(A)+ RNA. These findings demonstrate that rat liver and kidney contain only a single 5'-deiodinase which manifests either high or low Km activity depending on the reduced thiol cofactor utilized in the reaction.