W e studied the distribution of 5-hydroxytryptamine– (5-HT-) containing cells in the guinea pig pancreas and examined the effects of 5-HT on fluid secretion by interlobular pancreatic ducts. The 5-HT–immunoreactive cells with morphological characteristics of enterochromaffin (EC) cells were scattered throughout the duct system and were enriched in islets of Langerhans. The fluid secretory rate in the isolated interlobular ducts was measured by videomicroscopy. Basolateral applications of 5-HT strongly but reversibly reduced HCO₃-dependent, as well as secretin- and acetylcholine- (ACh-) stimulated, fluid secretion, whereas 5-HT applied into the lumen had no such effects. Secretin-stimulated fluid secretion could be inhibited by a 5-HT₃ receptor agonist, but not by agonists of the 5-HT₁, 5-HT₂, or 5-HT₄ receptors. Under the stimulation with secretin, 5-HT decreased the intracellular pH (pH_i) and reduced the rate of pH_i recovery after acid loading with NH₄⁺, suggesting that 5-HT inhibits the intracellular accumulation of HCO₃^–. The elevation of intraductal pressure in vivo reduced secretin-stimulated fluid secretion, an effect that could be attenuated by a 5-HT₃ receptor antagonist. Thus, 5-HT, acting through basolateral 5-HT₃ receptors, strongly inhibits spontaneous, secretin-, and ACh-stimulated fluid secretion by guinea pig pancreatic ducts. 5-HT released from pancreatic ductal EC cells on elevation of the intraductal pressure may regulate fluid secretion of neighboring duct cells in a paracrine fashion.