5-Hydroxytryptamine (serotonin, 5-HT) is released from the gastrointestinal tract by vagal stimulation. This biogenic amine produces many alterations in gastric functional parameters, including inhibition of gastric acid secretion. This study was designed to characterize the 5-HT receptor subtype modulating gastric acid secretion. In urethane-anesthetized acute gastric fistula rats, systemic 5-HT (3.5 mumol/kg i.v.) inhibited acid output stimulated by pentagastrin infusion by 58%. Close gastric intra-arterial (i.a.) injection of methysergide, methiothepin, metergoline or spiperone but not tropisetron, renzapride or ritanserin was effective in reversing 5-HT-induced inhibition of acid secretion. Close i.a. administration of the potent 5-HT1A/1B antagonist 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine displayed partial agonist properties in this model, but did not antagonize 5-HT-induced inhibition of acid output. In a study of 5-HT agonists given close i.a. to the gastric circulation, 5-HT (0.88 mumol/kg) inhibited acid secretion by 48%. A 3-fold higher dose (2.6 mumol/kg) of the general 5-HT1 agonist, 5-carboxamidotryptamine (5-CT), was needed to inhibit acid secretion significantly. In contrast, neither the selective 5-HT1A agonist (+-)-8-hydroxy-2-(n-dipropylamino)-tetralin nor 5-HT2 agonist (+-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (0.88 or 2.6 mumol/kg) attenuated gastric acid secretion. Thus, the data suggest that the site mediating inhibition of acid secretion by exogenous 5-HT belongs to the 5-HT1 family, but may not be of the 5-HT1A subtype.