Pituitary adenomas were obtained from eight of nine patients with Cushing's disease, and the surrounding tissues as well were obtained from six of nine patients. ACTH,β- lipotropin (0-LPH),β-endorphin, GH, TSH, LH, and PRL concentrations in these tissues were determined by RIA. Immunoreactive ACTH and β-endorphin (β-endorphin β-LPH) were present in high concentrations in all adenomas, and low concentrations were found in the surrounding tissues, except for one patient. As compared to levels seen in normal pituitary tissue, the GH concentration in the surrounding tissues was suppressed in five of six cases. TSH and LH concentrations were suppressed in four and three cases, respectively. The PRL concentration was not suppressed in any of the six patients studied. These four hormones were not detected in any adenoma. Plasma GH, TSH, and LH responses to various stimuli which were suppressed preoperatively returned to normal in most of the patients after adenomectomy. Basal plasma cortisol concentrations were normal or subnormal and were suppressed by the administration of 1 mg dexamethasone after adenomectomy, in contrast to the lack of such suppression preoperatively. ACTH and β-endorphin secretion were stimulated by lysine-8-vasopressin and suppressed by dexamethasone and cyproheptadine in vitro.