Peptide YY (PYY) and neuropeptide Y (NPY), infused into the quiescent isolated perfused canine ileum, dose-dependently increased phasic activity of the circular muscle and decreased tonic output of immunoreactive vasoactive intestinal peptide (VIP) in the venous effluent. The contractions subsided before the prolonged inhibition of VIP output. Motor excitation by PYY, an abundant neuropeptide in this tissue, was reduced by blockade of muscarinic or nicotinic receptors, or inhibition of nitric oxide (NO) synthase, despite continued inhibition of VIP output. A combination of atropine and hexamethonium eliminated the PYY-induced decrement in VIP output and left motor excitation unchanged. Blockade of NO synthase eliminated motility increases under these conditions. Intracellular microelectrode recordings of myenteric plexus-free circular muscle strips found no effect of NPY on the resting membrane potential, or on the field stimulation-induced inhibitory junction potential. Inhibition of VIP release plays no essential role in changing motility. These results suggest that PYY/NPY induce motility by stimulating release of acetylcholine and inhibiting NO release at a locus proximal to but not on nerve terminals.