Diazepam binding inhibitor (DBI) is a 9-kDa polypeptide that was initially isolated from rat brain and subsequently found to be present in several peripheral tissues. DBI is particularly abundant in steroidogenic tissues, such as the adrenal glands and testes, which also contain a high concentration of peripheral/mitochondrial benzodiazepine receptors (MBRs). Because occupancy of adrenal MBRs with DBI results in increased steroidogenesis, we have investigated the relation between ACTH, DBI, and the MBR in the rat adrenal glands. Evidence presented here indicates that both the amount of DBI and its rate of synthesis in the adrenal cortex are under the control of ACTH. Seven and 9 days after hypophysectomy, the amount of DBI-like immunoreactivity (DBI-LI) in rat adrenal glands decreased dramatically from approximately 80 to 15 ng/mg tissue. The administration of single dose of ACTH (ACTH residues 1-39; 200 mU/kg, iv) or repeated doses of ACTH-R (ACTH in saline containing 16% gelatin; 15 U/kg, sc, twice daily) reduced the decrease in adrenal DBI-LI caused by hypophysectomy. In hypophysectomized rats (7 days after hypophysectomy) the increases in both adrenal DBI-LI and plasma corticosterone induced by ACTH 1 h after a single injection (200 mU/kg, iv) were inhibited by injection of cycloheximide (40 mg/kg, ip) 10 min after ACTH. However, cycloheximide at this dose had no effect on the ACTH-induced increase in adrenal cAMP concentration or the number of affinity of MBRs for 4'-[3H]chlorodiazepam.