Abstract The role of vasoactive intestinal peptide (VIP) was investigated when mucosal stroking and 5-hydroxytryptamine (5-HT) were used to activate neural reflexes that stimulate chloride secretion in the guinea pig colon. Muscle-stripped segments of colon containing intact submucosal ganglia without myenteric ganglia were set up in modified flux chambers in order to record short-circuit current (Isc). Mucosal stroking with a brush for 1 s or a pulse of 5-HT (injection of 15 μl of 100 μM 5-HT into 1.5 ml of mucosal solution) caused an increase in Isc that was reduced by the VIP antagonist, neurotensin 6–11-VIP 7–28, in a concentration-dependent manner. The Isc responses to mucosal stroking and a 5-HT pulse were reduced by 53% and 58%, respectively, by 2 μM neurotensin 6–11-VIP 7–28. The residual Isc response in the presence of neurotensin 6–11-VIP 7–28 was abolished by atropine. Blockade of 5-HT 1P receptors on submucosal afferent neurons decreased Isc responses to stroking or a 5-HT pulse. The residual Isc response after 5-HT 1P receptors were blocked was reduced by only 11–14% by neurotensin 6–11-VIP 7–28. In the presence of blockade of both 5-HT 1P and VIP receptors, atropine abolished the Isc response to both stimuli. The observations suggest that the neural circuitry activated by stroking includes at least two independent pathways. One pathway contains VIP neurons which receive inputs directly or indirectly from 5-HT 1P receptor-containing afferents. A second pathway involves muscarinic cholinergic transmission that is independent of 5-HT 1P and VIP receptor activation.