Orexins A and B are two hypothalamic peptides that increase food intake and body weight and probably play a role in the sleep regulation. They act through two subtypes of G protein-coupled receptors, called OX1-R and OX2-R. OX1-R selectively binds orexin-A, whereas OX2-R is nonselective for both orexins. Orexins did not affect the in vitro secretion of either catecholamine or aldosterone from human adrenals. Conversely, orexin A, but not orexin B, concentration dependently increased basal cortisol secretion from dispersed adrenocortical cells; the maximal effective concentration was 10(-8) mol/L. Orexin A (10(-8) mol/L) enhanced the cortisol response to maximal effective concentrations (10(-9) mol/L) of angiotensin II and endothelin-1, but only to low concentrations of ACTH (10(-12)/10(-11) mol/L). Orexin A (10(-8) mol/L) increased basal cAMP release by dispersed adrenocortical cells, and the effect was blocked by the adenylate cyclase inhibitor SQ-22536. The cortisol response to 10(-8) mol/L orexin A was unaffected by the ACTH receptor antagonist corticotropin-inhibiting peptide, but was abolished by either SQ-22536 or the protein kinase A inhibitor H-89. RT-PCR demonstrated high levels of OX1-R messenger ribonucleic acid and very low levels of OX2-R messenger ribonucleic acid in human adrenal zona fasciculata-reticularis and adrenal medulla. Collectively, our findings suggest that orexins selectively stimulate glucocorticoid secretion from human adrenocortical cells, acting through OX1-R coupled with the adenylate cyclase-dependent signaling pathway.