This report describes the application of a sensitive, specific, and reproducible RIA for diiodotyrosine (DIT) in human serum and metabolic studies on the source and kinetics of circulating DIT. Interference by cross-reactivity of T4 and other analogs was completely eliminated by isolation of DIT from serum with an efficient preparative immunoprecipitation technique. Mean (+/- SD) serum DIT levels were 161 +/- 133 pmol/liter (7.0 ng/100 ml) in 41 normal subjects, 64 +/- 30 pmol/liter in 46 pregnant women, 241 +/- 83 pmol/liter in the cord serum of 48 newborn infants, 542 +/- 494 pmol/liter in 22 hyperthyroid patients, and 101 +/- 71 pmol/liter in 15 hypothyroid patients. Mean values in pregnant, newborn and hyperthyroid subjects were significantly different from the normal mean. Very low DIT serum levels were found in four athyreotic patients during oral T4 substitution therapy, indicating that little DIT is formed by peripheral T4 degradation. In five normal subjects who received a single oral dose of 3 mg T4, serum DIT remained unchanged in one case and decreased in four cases. Radioimmunological measurements of DIT elimination from serum after the iv injection of 1 mg DIT in two normal volunteers gave MCRs of 103 and 133 liters/day and an average extrathyroidal DIT turnover rate of 19 nmol/day (8.2 microgram/day). These data indicate that circulating DIT arises predominantly from the thyroid, suggesting that peripheral formation of DIT is a minor metabolic pathway in the human.