A clincally useful and reproducible radioimmunoassay for human homologous prolactin, established with commercially available reagents, was studied and validated. We present detailed conditions for iodination and purification of labeled prolactin and the optimal conditions for the assay. By the method, we found values (microgram/liter) as follows for serum prolactin: normal men. 8.9 +/- 5.2 (mean +/- SD); normal women, 11.8 +/- 5.5; normal women taking contraceptive pills, 9.2 +/- 5.0; pregnant women in the third trimester, 188 +/- 69.5; patients with various diseases other than of the hypothalamic-pituitary axis, 9.3 +/- 6.3; in some patients with amenorrhea and galactorrhea of diverse origin, 78.2 +/- 87.4; and in some patients with surgically proven pituitary tumor, 1414 +/- 1980. Results under provocative testing are also presented for a patient with normal hypothalamic-pituitary function.