Sulfation is an important component of human thyroid hormone metabolism. The role of the human sulfotransferase 1C1 (SULT1C1) is not known. Because SULT1C1 is present in the adult thyroid, intra-thyroidal sulfation of thyroid hormones and their metabolites might occur. We tested this hypothesis by determining the ability of recombinant human SULT1C1 to catalyze iodothyronine sulfation. Apparent K(m) values for 3,3',5-triiodothyronine (T(3)), 3, 3'-diiodothyronine (3,3'-T(2)), 3',5',3-triiodothyronine (rT(3)), and 3,3',5,5'-tetraiodothyronine (T(4)) with SULT1C1 were 28.7, 10.3, 10.2, and 59.3 microM, respectively. Thermal stability and responses to inhibitors also were tested with T(3) as the substrate. Enzyme aliquots were measured simultaneously to determine SULT1C1 substrate preferences at optimal iodothyronine concentrations. SULT1C1 activity obtained with T(3) was used as 100%, and the activities with 3,3'-T(2), rT(3), T(4), and 3,5-diiodothyronine (3, 5-T(2)) were 614, 314, 25, and 4%, respectively. We report for the first time the characterization of human SULT1C1 with T(3) and the preferences of the enzyme for various iodothyronines. The presence of SULT1C1 in the adult thyroid gland raises the possibilities that the enzyme can contribute to intraglandular thyroid hormone processing and iodide reutilization.