Diazepam-binding inhibitor (DBI) is a 9-kDa polypeptide that colocalizes in glial, adrenocortical, and Leydig cells with the mitochondrial DBI receptor (MDR). By binding with high affinity to the MDR, DBI and one of its processing products--DBI-(17-50)--regulate pregnenolone synthesis and have been suggested to participate in the immediate activation of adrenal steroidogenesis by adrenocorticotropic hormone (ACTH). In adrenals of hypophysectomized rats (1 day after surgery), ACTH failed to acutely affect the amount of adrenal DBI and the density of MDR but increased the rate of DBI processing, as determined by the HPLC profile of DBI-(17-50)-like immunoreactivity. The similar latency times for this effect and for ACTH stimulation of adrenal steroidogenesis suggest that the two processes are related. The ACTH-induced increase in both adrenal steroidogenesis and rate of DBI processing were completely inhibited by cycloheximide; this result suggests the requirement for the de novo synthesis of a protein with a short half-life, probably an endopeptidase. This enzyme, under the influence of ACTH, may activate formation of a DBI-processing product that stimulates steroidogenesis via the MDR. In support of this hypothesis is the demonstration that in hypophysectomized rats the MDR antagonist PK 11195 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide completely inhibited the adrenal steroidogenesis stimulated by ACTH and by the high-affinity MDR ligand 4'-chlorodiazepam.