The rate-limiting step in the biosynthesis of steroids is the transport of the substrate cholesterol from the outer to the inner mitochondrial membrane, where cholesterol is metabolized to pregnenolone. This transport is markedly stimulated by the action of hormones, such as adrenocorticotropic hormone (ACTH) and luteinizing hormone (LH) for adrenocortical and testicular Leydig cells, respectively. Recently, it was demonstrated that the peripheral-type or mitochondrial benzodiazepine receptor, abundant in steroidogenic tissues, is involved in the regulation of steroid biosynthesis. In search for an endogenous ligand for mitochondrial benzodiazepine receptors, regulating steroidogenesis, the effects of Diazepam Binding Inhibitor (DBI) were studied. The model systems used were the Y-1 adrenocortical and the MA-10 Leydig cell lines, previously shown to be valid steroidogenic models. Both cell lines contain significant levels of immunoreactive DBI. Purified DBI from rat brain, at high nanomolar concentrations, increased formation of pregnenolone, when added to mitochondrial preparations of both cell types; but at concentrations of DBI above 1 microM, a decrease in the stimulation was observed. Flunitrazepam, a benzodiazepine which binds to mitochondrial benzodiazepine receptors, with high nanomolar affinity, inhibited the stimulatory action of DBI on the formation of mitochondrial pregnenolone, indicating that DBI exerts its stimulatory effects through an action on mitochondrial benzodiazepine receptors. In order to determine the biologically active amino acid sequence in the DBI molecule, various fragments of DBI were synthesized and tested; also, peptides structurally unrelated to DBI were tested.(ABSTRACT TRUNCATED AT 250 WORDS)