5-Hydroxytryptamine (serotonin, 5-HT) is a hormone and neurotransmitter regulating gastrointestinal functions. 5-HT receptors are widely distributed in gastrointestinal mucosa and the enteric nervous system. Duodenal acidification stimulates not only the release of both 5-HT and secretin but also pancreatic exocrine secretion. We investigated the effect of 5-HT receptor antagonists on the release of secretin and pancreatic secretion of water and bicarbonate induced by duodenal acidification in anesthetized rats. Both the 5-HT₂ receptor antagonist ketanserin and the 5-HT₃ receptor antagonist ondansetron at 1-100 µg/kg dose-dependently inhibited acid-induced increases in plasma secretin concentration and pancreatic exocrine secretion. Neither the 5-HT₁ receptor antagonists pindolol and 5-HTP-DP nor the 5-HT₄ receptor antagonist SDZ-205,557 affected acid-evoked release of secretin or pancreatic secretion. None of the 5-HT receptor antagonists affected basal pancreatic secretion or plasma secretin concentration. Ketanserin or ondansetron at 10 µg/kg or a combination of both suppressed the pancreatic secretion in response to intravenous secretin at 2.5 and 5 pmol · kg¹ · h¹ by 55-75%, but not at 10 pmol · kg¹ · h¹. Atropine (50 µg/kg) significantly attenuated the inhibitory effect of ketanserin on pancreatic secretion but not on the release of secretin. These observations suggest that 5-HT₂ and 5-HT₃ receptors mediate duodenal acidification-induced release of secretin and pancreatic secretion of fluid and bicarbonate. Also, regulation of pancreatic exocrine secretion through 5-HT₂ receptors may involve a cholinergic pathway in the rat.