We have employed a new in vitro assay for study of the T3 sulfation activity in rat tissues. The assay measures by RIA the generation of T3 sulfate (T3S) during incubation of T3 with cytosol of rat tissues as the source of phenol sulfotransferase(s) and 3-phosphoadenosine-5'-phosphosulfate as the sulfate donor. The conversion of T3 to T3S proceeded rapidly for 30 min at 37 C, and the optimal pH of the reaction was 8.0. Heating the cytosol at 44 C for 15 min decreased T3S production to 63% of its value at 37 C. T3 sulfation activity was plentiful in rat liver, brain, and kidney, but little activity was demonstrable in other tissues. The Km and maximum velocity of the hepatic conversion of T3 to T3S were 114 microM and 159 pmol/mg protein.h, respectively. There was a marked inhibition of the conversion of T3 to T3S with salicylamide, 3'-monoiodothyronine, thyronine, and rT3; the IC50 of these inhibitors approximated 15, less than 0.1, 9.5, and 43 microM, respectively. On day 17 of gestation, the T3 to T3S conversion activity was more abundant in fetal skin than in other fetal tissues. However, the activity decreased in fetal skin while it increased in fetal liver, kidney, and brain nearer to term on day 20. Placenta demonstrated lower T3 to T3S conversion activity than several fetal or maternal tissues. There was no effect of hypothyroidism or hyperthyroidism on T3 sulfation activity. We conclude that T3 sulfation activity in the rat is 1) most abundant in liver, kidney, and brain tissues of the adult; 2) inhibited more avidly by 3'-monoiodothyronine than other thyronines; 3) very abundant in fetal skin early in gestation; and 4) little affected by the thyroidal status of the animal.