Orexin-A is a neuropeptide consisting 33 amino acids with two intrachain disulfide bonds, namely Cys6-Cys12 and Cys7-Cys14, and is a potent stimulator of food consumption and gastric acid secretion. In contrast, orexin-B, a peptide containing 28 amino acids without disulfide bond, which has no stimulatory action of gastric acid. The objective of the present study was to characterize the receptor-mediated mechanism of orexin-A-induced stimulation of gastric acid secretion using orexin-Arelated peptides with modification of disulfide bonds. Intracisternal injection of orexin-A but not orexin-B or orexin-A (15-33) that does not contain both disulfide bonds stimulated gastric acid secretion in pylorusligated conscious rats. The ability of the stimulation of gastric acid output was less in three alanine-substituted orexin-A, [Ala^(6, 12)]orexin-A, [Ala^(7, 14)]orexin-A and [Ala^(6, 7, 12, 14)]orexin-A, than orexin-A. Orexins-induced calcium increase was measured in CHO-K1 cells expressing OX_1R or OX_2R. Orexin-A induced a transient increase in [Ca^(2+)]I in CHO-K1/OX_1R cells in a dose-dependent manner. EC_50 values for OX_1R of orexin-A, orexin-B or orexin-A (15-33) was 0.068, 0.69 or 4.1 nM, respectively, suggesting that peptides containing no disulfide bonds have lower potency for the receptor. Agonistic activity for OX1R of the three orexin-A analogues with modification of one or both disulfide bonds was significantly reduced as compared with that of orexin-A. EC_50 values for OX_2R of orexin-A and orexin-B was almost equal but potency for the receptor of orexin-A (15-33) and three alanine substituted orexin-A was less than that of orexin-A. A significant inverse relationship between gastric acid output and EC_50 values for OX_1R but not OX_2R was observed. These results suggested that the orexin-A-induced acid stimulation requires OX_1R activation and that disulfide bonds in orexin-A may have a key role in the receptor activation.