The physiological role of GRP and of its cognate receptors in regulating the intestinal peristaltic reflex was examined in a three compartment flat-sheet preparation of rat colon. Mucosal stimulation applied to the central compartment at high but not low levels of intensity induced GRP release in the caudad compartment where descending relaxation was measured but not into the ascending compartment where ascending contraction was measured, or into the central compartment where the stimuli were applied. The selective GRP (BB₂) receptor antagonist, [D-Phe⁶, des-Met¹⁴] bombesin _(6-14) , inhibited descending relaxation and VIP release in the caudad compartment induced by high but not low levels of stimulation applied to the mucosa in the central compartment. The selective NMB (BB₁) receptor antagonist, BIM-23127, had no effect on descending relaxation or VIP release. Neither the BB₁ nor the BB₂ antagonist had any effect on ascending contraction or SP release in the orad compartment. Consistent with the effects of the antagonists on the peristaltic reflex, the BB₂ antagonist but not the BB₁ antagonist decreased the velocity of propulsion of artificial fecal pellets through isolated segments of guinea pig distal colon. The results indicate that GRP is selectively released from myenteric neurons in descending pathways during the peristaltic reflex and that it acts via BB₂ receptors to augment the descending phase of the peristaltic reflex and propulsion.