ACTH-producing pituitary adenomas obtained from two patients with Cushing's disease were maintained in organ culture for 2 weeks. After 4-6 days of incubation, hydrocortisone (0.1, 1, and 5 mg/ml) was added to the culture medium. Addition of cortisol to the media in concentrations which generally inhibit the ACTH secretion in vivo in patients with Cushing's disease (1 mg/ml) failed to affect the ACTH secretion in vitro. A decrease of ACTH secretion occurred only with 5 mg/ml after 5 days of incubation. A similar effect on growth hormone (GH) release was seen when cortisol (10 mg/ml) was added to cultures from GH-producing pituitary adenoma. No ultrastructural changes were found in cultured cells that could be attributed to the addition of 0.1 mg/ml cortisol in the culture medium. With higher concentrations, (1 mg/ml and 5 mg/ml) minor ultrastructural changes were suspected. Our present findings implicate the hypothalamus as the target area for the feedback control of ACTH in Cushing's disease but could not reveal whether or not the primary lesion (i.e. the tumor) was of pituitary or hypothalamic origin. Our organ culture system appears to be a suitable model for investigating possible ACTH-regulating factors.