These studies were performed to determine how bilateral adrenal hypertrophy persists despite normal resting ACTH levels in male rats for weeks after a 3-day course of treatment with cyanoketone (CK). This drug blocks corticosterone synthesis by binding to the enzyme 3 beta-ol-dehydrogenase-delta 5, delta 4-isomerase (EC 1.1.1.51). Although plasma ACTH levels were significantly elevated at all times during the course of treatment with CK, ACTH and corticosterone levels of CK-treated rats were normal thereafter compared to those in control rats killed in the morning. Despite these normal ACTH levels, adrenal weight remained elevated (by 100%) for at least 14 days. We determined that increased ACTH secretion was required to initiate and maintain the adrenal growth response to CK. Rats pretreated with hypophysectomy or dexamethasone (1 mg/kg) did not respond with increases in adrenal weight after CK treatment. Sustained elevations in ACTH during CK treatment were required for increased adrenal weight, because rats treated with dexamethasone 3 h after CK injection did not have enlarged adrenals at 24 h. Increased adrenal weight was not sustained after removal of supranormal levels of ACTH achieved by infusion, suggesting that after adrenal hypertrophy is achieved by ACTH, elevated ACTH levels are also required to maintain adrenal enlargement. Finally, ACTH and corticosterone levels were measured in the evening (at the peak of the diurnal rhythm in the adrenocortical system of rats) in CK-treated rats. Evening ACTH levels were significantly elevated in CK-treated rats compared to those in controls 7, 10, and 14 days after the onset of 3 days of treatment with CK; however, corticosterone levels were normal. The effects of CK on the adrenocortical system were exerted via the adrenal, since adrenalectomy normalized the amplitude of the diurnal rhythm of ACTH in CK-treated rats compared to that in adrenalectomized controls. We conclude 1) that adrenal hypertrophy after CK is maintained by increased ACTH secretion which occurs daily in the evening; and 2) that the results provide evidence for a daily reset of the corticosteroid feedback sensor in the adrenocortical system. This conclusion arises from the findings that there is a normal rhythm in corticosterone levels in CK-treated rats and that morning ACTH levels are normal although evening ACTH levels are significantly elevated.