Cells isolated from 24 autopsied adult human pituitary glands by enzymatic dissociation and Percoll density gradient methods were maintained in culture for up to 10 months. Using rabbit antisera specific for individual pituitary hormones and immunoperoxidase staining, we found a large number of cultured pituitary cells to react positively to growth hormone, prolactin, adrenocorticotropin, follicle-stimulating hormone, luteinizing hormone, and thyrotropin immunoreactions. Electron microscopic examination of cultured pituitary cells demonstrated the occurrence of secretory granules in cytoplasm characteristic of in vivo human pituitary cells. The hormone secretion in culture by these pituitary cells was confirmed by the detection of prolactin, thyrotropin, and follicle-stimulating hormone in the culture medium by radioimmunoassay.