Sulfation appears to be an important pathway for the reversible inactivation of thyroid hormone during fetal development. The rat is an often used animal model to study the regulation of fetal thyroid hormone status. The present study was done to determine which sulfotransferases are important for iodothyronine sulfation in the rat, using radioactive T4, T3, rT3, and 3,3\'-T2 as substrates, 3\'-phosphoadenosine-5\'-phosphosulfate (PAPS) as cofactor, and rat liver, kidney and brain cytosol, and recombinant rat SULT1A1, 1B1, 1C1, 1E1, 2A1, 2A2 and 2A3 as enzymes. Recombinant rat SULT1A1, 1E1, 2A1, 2A2 and 2A3 failed to catalyze iodothyronine sulfation. For all tissue sulfotransferases and for rSULT1B1 and rSULT1C1, 3,3\'-T2 was by far the preferred substrate. Apparent K_m values for 3,3\'-T2 amounted to 1.9 µM in male liver, 4.4 µM in female liver, 0.76 µM in male kidney, 0.23 µM in male brain, 7.7 µM for SULT1B1, and 0.62 µM for SULT1C1, while apparent K_m values for PAPS showed less variation (2.0-6.9 µM). Sulfation of 3,3\'-T2 was inhibited dose-dependently by other iodothyronines, with similar structure-activity relationships for most enzymes except for the sulfotransferase activity in rat brain. The apparent K_m values of 3,3\'-T2 in liver cytosol were in between those determined for SULT1B1 and 1C1, supporting the importance of these enzymes for the sulfation of iodothyronines in rat liver, with a greater contribution of SULT1C1 in male than in female rat liver. The results further suggest that rSULT1C1 also contributes to iodothyronine sulfation in rat kidney, whereas other, yet unidentified forms appear more important for the sulfation of thyroid hormone in rat brain.