Background and Aims: Similar to cholecystokinin (CCK), non-CCK-dependent duodenal factors stimulate vagal mucosal afferent fibers to mediate pancreatic enzyme secretion via a common cholinergic pathway. We tested the hypothesis that 5-hydroxytryptamine (5-HT) released from enterochromaffin (EC) cells plays an important role in the transduction of luminal information to the central nervous system via vagal afferent fibers to mediate pancreatic secretion. Methods: Pancreatic secretions were examined in conscious rats after intragastric administration of chopped rodent chow in the presence and absence of CCK or 5-HT3 and 5-HT2 antagonists. Pancreatic responses to intraduodenal administration of maltose, hyperosmolar NaCl, and light mucosal stroking were examined in rats pretreated with various pharmacological antagonists or after surgical or chemical ablation of vagal and 5-HT neural pathways. Results: Administration of L364,718 inhibited 54% of pancreatic protein secretion evoked by intragastric administration of rodent chow. L364,714 and ICS 205-930, a 5-HT3 antagonist, combined produced a 94% inhibition. Vagal afferent rootlet section eliminated pancreatic secretions evoked by intraduodenal stimuli, p-Chlorophenylalanine, a 5-HT synthesis inhibitor, but not 5,7-hydroxytryptamine, a 5-HT neurotoxin, also eliminated the pancreatic response to these luminal stimuli. The 5-HT3 antagonist markedly inhibited pancreatic secretion induced by maltose and hyperosmolar NaCl. 5-HT2 and 5-HT3 antagonists combined inhibited the pancreatic response to light stroking of the mucosa. Conclusions: Luminal factors such as osmolality, disaccharides, and mechanical stimulation stimulated pancreatic secretion via intestinal vagal mucosal afferent fibers. It is likely that 5-HT originating from intestinal EC cells activated 5-HT3 and 5- HT2 receptors on vagal afferent fibers to mediate luminal factor-stimulated pancreatic secretion.