Neural reflex pathways activated in response to mucosal stroking were investigated in segments of distal colon from guinea pigs. Stroking the mucosal surface of whole thickness or muscle-stripped segments with a brush at 1/s evoked an increase in short-circuit current (Isc) whose duration and amplitude were dependent on the number of strokes. The increase in Isc induced by mucosal stroking was virtually abolished by inhibitors of chloride secretion and by tetrodotoxin. The response was reduced by atropine, renzapride, and N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide but not by mecamylamine, ketanserin, tropisetron, or SDZ-205-557. Mucosal application of 5-hydroxytryptamine and 5-hydroxyindalpine increased Isc, which was attenuated by tetrodotoxin and renzapride. The results suggest that mucosal stroking evokes chloride secretion by activating neural reflex pathways utilizing serotoninergic receptors, of the 5-HT1P class, as well as muscarinic receptors within the submucosal plexus.