Sulfated iodothyronines including T4-sulfate (T4S) and T3-sulfate (T3S) have been identified in human serum and amniotic fluid. Little is known, however, about the existence of sulfate conjugation of reverse T3 (rT3S) in man. In this report, we employed a novel, sensitive, and specific rT3S RIA to address this question. The rabbit antiserum to rT3S was highly specific; T4, T3, rT3, and 3,3'-T2 showed less than 0.002% cross-reaction with the antiserum. Only T4S and T3S cross-reacted significantly (0.3% and 0.01%, respectively); other analogs cross-reacted less than 0.0001%. The detection threshold of the RIA was 14 pmol/L (1.0 ng/dL). The mean serum rT3S concentration (pmol/L) was 40 in euthyroid subjects. Values were similar in hypothyroid patients (38) and pregnant women (52) but significantly (P < 0.01) elevated to 176 in hyperthyroid patient, 74 in patients with nonthyroid illnesses, and 684 in cord sera of newborns. Serum rT3S increased significantly in hyperthyroid patients 1 day after administration of 1 g sodium ipodate orally. Reverse T3S was detected consistently in amniotic fluid at 14 to 22 weeks of gestation and showed a marked rise 1-3 weeks after intraamniotic administration of 500-1000 micrograms T4. The various data suggest that: (1) rT3S is a normal component of human serum and amniotic fluid; (2) it is derived from metabolism of T4 or rT3; (3) circulating rT3S increases in hyperthyroidism and in circumstances where type I 5'-monodeiodinating activity is low, e.g. nonthyroid illnesses, fetal life, and after administration of ipodate.