Serum concentrations of dehydroepiandrosterone sulfate (DHEA-S) were measured in patients with hyperadrenocorticism. When compared to normal subjects of corresponding age, serum DHEA-S levels were normal or elevated in 37 patients with Cushing's disease. In contrast, DHEA-S levels were significantly lower than those of normal subjects in all 28 patients with hyperadrenocorticism due to benign adrenocortical adenoma, suggesting that ACTH is the major determinant of DHEA-S secretion and that determination of serum DHEA-S concentrations is useful in the biochemical differential diagnosis of the etiology of Cushing's syndrome. In six patients with adrenocortical adenoma, the recovery of suppressed DHEA-S secretion after removal of the adrenal gland affected by a tumor was studied. Serum cortisol levels normalized by the end of the second year after unilateral adrenalectomy, while DHEA-S levels remained low for at least 2 succeeding yr. The results suggest that deficient ACTH secretion may result in a greater and longer lasting loss in the ability of the adrenal cortex to secrete androgens than in the ability to secrete cortisol.