Possible interactions of ACTH-like peptides with melatonin regulation of central-type benzodiazepine (BNZ) receptors have been studied by means of high-affinity 3H-flunitrazepam binding to rat cerebral cortex membrane preparations. Intracerebroventricular injections of melatonin produce a dose-dependent increase in Bmax in pinealectomized rats, without changes in KD. Analogous effects were obtained after intracerebroventricular injection of melatonin in adrenalectomized and in adrenalectomized plus pinealectomized rats, which indicated the lack of participation of adrenal steroids in this response. Moreover, intracerebroventricular injection of ACTH1-10 induced a similar dose-dependent increased Bmax in sham-operated animals, whereas pinealectomy, but not adrenalectomy, partially counteracted this effect of ACTH1-10 administration. Besides, simultaneous injection of ACTh1-10 plus melatonin reverses the effects of pinealectomy, resulting in an additive effect of both compounds on Bmax. The response obtained when using ACTh4-10 was somewhat different, because no dose response was obtained in any experiment. Although lack of endogenous melatonin partially reduced the increasing effect of ACTH4-10 on Bmax, there were no additive effects at the different doses used. The results strongly suggest that ACTH-like peptides, in addition to melatonin, play a role in regulating central-type rat BNZ receptors.