The biosynthesis of growth hormone and prolactin by human pituitary glands in vitro was studied by incubation with l-4,5-leucine-3H. The histology of 2 glands, one from a male and one from a female, was normal; 3 others were pituitary adenomas from patients with various endocrine disturbances: acromegaly, galactorrhea and amenorrhea. Immunoprecipitation studies were carried out on the incubation media using antiserum to ovine prolactin (anti-OPr) and to human growth hormone (anti-HGH). In the normal pituitary from the female and the adenoma from the patient with galactorrhea, anti-OPr precipitated 3 and 30 times more radioactive proteins in the respective media than did anti-HGH. By contrast, in the pituitary of the male and the adenoma of the acromegalic, anti-HGH precipitated 3 and 30 times more radioactive proteins than did anti-OPr in the respective media. In the adenoma from the amenorrheic patient (a chromophobe adenoma) both anti-OPr and anti-HGH precipitated only 10% of the radioactive proteins in the medium. HGH and human prolactin (HPr) concentrations in the incubation media were measured by radioimmunoassay. In the 2 normal pituitaries, the HPr: HGH ratio showed a marked variation with sex, being 10 times higher in the female. Among the adenomas, HPr was detectable only in the incubation medium of the adenoma from the galactorrheic patient. These findings correlate well with the results which could be anticipated from the clinical status of the patients and provide strong evidence that, in man, HPr and HGH are separate hormones which can be clearly distinguished immunologically.