Neurokinins regulate gastrointestinal motility by interacting with receptors on both muscle layers and on myenteric plexus neurons. To determine if specific neurokinin (NK) receptor agonists can mediate inhibitory effects on myenteric neurons, we studied the effect of the NK-1 agonist substance P methylester (SPME) and the putative endogenous NK-2 receptor ligand neurokinin A (NKA) on [3H]acetylcholine [( 3H]ACh) release induced by electrical field stimulation from muscle strips cut from the canine gastric antrum. SPME but not NKA caused a dose-dependent inhibition of stimulated [3H]ACh release in tissues containing the myenteric plexus. The inhibition was not seen in longitudinal muscle without myenteric plexus. Pretreatment of tissues with indomethacin or antiserum to vasoactive intestinal polypeptide (VIP) but not naloxone or adrenergic or cholingergic blockade abolished the SPME-induced inhibition. Exogenous VIP stimulated the release of prostaglandin E2 (PGE2) from full thickness strips, and both VIP and PGE2 inhibited [3H]ACh release induced by electrical depolarization. These findings suggest that NK-1 receptor agonists can selectively inhibit stimulated [3H]ACh release and that this inhibition may involve the release of VIP and PGE2 from neurons within the myenteric plexus.