The pineal hormone melatonin participates in circadian, seasonal, and reproductive physiology. The presence of melatonin binding sites in human brain and peripheral tissues is well documented. However, in the mammalian adrenal gland, low-affinity melatonin binding sites have been detected only in the rat by some but not all authors. Conflicting evidence for a regulatory role of melatonin on adrenal cortisol production, prompted us to investigate this possibility in a New World primate, the capuchin monkey. Expression of melatonin receptors in the adrenal cortex was demonstrated through pharmacological characterization and autoradiographic localization of 2-[125I]iodomelatonin binding sites (dissociation constant = 96.9 +/- 15 pM; maximal binding capacity = 3.8 +/- 0.4 fmol/mg protein). The mt1 identity of these receptors was established by cDNA sequencing. Melatonin treatment of dispersed cells and explants from adrenal gland did not affect basal cortisol production. However, cortisol production stimulated by 100 nM ACTH was significantly inhibited by low melatonin concentrations (0.1-100 nM); this inhibitory effect was reversed by the mt1/MT2 melatonin antagonist luzindole. Melatonin also inhibited dibutyril-cAMP-stimulated cortisol production, suggesting that melatonin acts through a cAMP-independent signaling pathway. The present data demonstrate that the primate adrenal gland cortex expresses functional mt1 melatonin receptors and shows that melatonin inhibits ACTH-stimulated cortisol production.