We have developed a sensitive homologous radioimmunoassay for human prolactin (hPRL). Prolactin isolated from human pituitary glands was used for generating antibodies in rabbits, for iodination, and for standards. The assay is highly specific for hPRL and measures concentrations of hPRL as low as 1 ng/ml of human plasma. In this assay, the plasma concentrations of hPRL in a group of randomly selected male and female individuals averaged 13 ± 4 (SD) and 14 ± 4 ng/ml, respectively. In contrast, hPRL in the pooled plasma of pregnant women assayed 80 ng/ml; a post-partum plasma measured 215 ng/ml. Of 6 patients with acromegaly, 5 possessed hPRL levels above 34 ng/ml, the average being 40 ± 9 ng/ml. Administration of TSH releasing hormone increased plasma hPRL concentrations by 4-fold in both a male and a female subject, while LH releasing hormone depressed them slightly. The concentration of hPRL in pituitary glands removed at autopsy was 1.5 μg/mg for a male and 1.3 μg/mg for a female. These values approximate those found in other mammals.