Previous studies have shown that the inner ring deiodination (IRD) of T₃ and the outer ring deiodination (ORD) of 3,3’-diiodothyronine are greatly enhanced by sulfate conjugation. This study was undertaken to evaluate the effect of sulfation on T₄ and rT₃ deiodination. Iodothyronine sulfate conjugates were chemically synthetized. Deiodination was studied by reaction of rat liver microsomes with unlabeled or outer ring ¹²⁵I-labeled sulfate conjugate at 37 C and pH 7.2 in the presence of 5 mM dithiothreitol. Products were analyzed by HPLC or after hydrolysis by specific RIAs. T₄ sulfate (T₄S) was rapidly degraded by IRD to rT₃S, with an apparent K_m of 0.3 μm and a maximum velocity (V_max) of 530 pmol/min · μg protein. The V_max to K_m ratio of T₄S IRD was increased 200-fold compared with that of T₄ IRD. However, formation of T₃S by ORD of T₄S could not be observed. The rT₃S formed was rapidly converted by ORD to 3,3’-T2 sulfate, with an apparent K_m of 0.06 μm and a V_max of 516 pmol/min mg protein. The enzymic mechanism of the IRD of T₄S was the same as that of the deiodination of nonsulfated iodothyronines, as shown by the kinetics of stimulation by dithiothreitol or inhibition by propylthiouracil. The IRD of T₄S and the ORD of rT₃ were equally affected by a number of competitive inhibitors, suggesting a single enzyme for the deiodination of native and sulfated iodothyronines. In conjunction with previous findings on the deiodination of T₃S, these results suggest that sulfation leads to a rapid and irreversible inactivation of thyroid hormone. (Endocrinology117: 8–12, 1985)