Rabbit antiserum raised against a prolactin-rich extract of human amniotic fluid was used to establish a double-antibody radioimmunoassay for human pituitary prolactin inplasma. The binding of 131I-labelIed human pituitary prolactin(Lewis) to the antiserum was inhibited by unlabelled prolactin, human chorionic gonadotrophin (HCG), human luteinizing hormone (HLH) and human follicle stimulating hormone (HFSH). Absorption of the antiserum with HCG rendered the assay specific for prolactin no cross-reaction being then observed with HLH, HFSH, human thyroid stimulating hormone, human growth hormone or human chorionic somatomammotrophin. The binding of 131I-labelled human pituitary prolactin (Friesen) to antibody was not affected by HCG. The concentration of prolactin in plasmas from normal males, pregnant females, non-pregnant females and in patients treated with phenothiazine was measured.