The present report describes the development of a radioimmunoassay for 3,3',5'-L-triiodothyronine (reverse T3) which is performed on unextracted serum. Utilizing this radioimmunoassay, 21 normal subjects had a mean (+/-SD) serum reverse T3 level of 60 +/- 12 ng/100 ml, 17 of 19 hyperthyroid patients had elevated serum reverse T3 levels, and 10 of 11 hypothyroid subjects had decreased serum reverse T3 concentrations. Thyroidal secretion of reverse T3 was assessed by measurements in samples obtained from the internal carotid artery and jugular vein of sheep following the administration of thyrotropin releasing hormone (TRH) or bovine thyrotropin (TSH). Reverse T3 levels were increased 45-60 min after TRH administration, but TSH administration produced inconsistent alterations in reverse T3, although 18 of 27 samples obtained after TSH injection were higher than their average respective baseline concentration and the mean peak reverse T3 level was 14% higher than baseline. Following TRH administration to 10 normal human subjects, mean serum reverse T3 levels significantly increased from 53.6 ng/100 ml to 56.3ng/100 ml (P less than .05). The thyroid gland content of reverse T3 in human autopsy material was 6.5 +/- 1.5 microng/g tissue. Both pregnancy and estrogen administration were associated with increases in serum reverse T3 concentrations presumably because of their ability to augment thyroxine binding globulin synthesis.