The physiological role of NPY and of specific NPY receptors in regulating the intestinal peristaltic reflex was examined in three compartment flat-sheet preparations of rat colon. Graded muscle stretch or mucosal stimulation applied to the central compartment inhibited NPY release in the orad compartment where ascending contraction was measured. NPY and the Y₁ receptor agonist, [Leu³¹, Pro³⁴]NPY inhibited, whereas the selective Y₁ receptor antagonist, BIBP 3226, augmented ascending contraction and SP release in the orad compartment induced by muscle stretch or mucosal stimulation. Neither agonist nor antagonist had any effect on descending relaxation or VIP release in the caudad compartment. The Y₂ receptor agonist, NPY_13-36 and antagonist, BIIE 0246 had no effect on peptide release or mechanical response. The results indicate that suppression of a tonic inhibitory influence of NPY neurons on excitatory neurotransmitter release contributes substantially to the orad contractile phase of the peristaltic reflex. The effect of NPY on neurotransmitter release is mediated by Y₁ receptors.