BACKGROUND Motilin is known to induce gastric phase III contractions via neural pathways in vivo, but the local mechanism of action is not clearly determined. METHODS An isolated perfused canine stomach was used to demonstrate the mechanism of motilin. Synthetic canine motilin at doses of 0.1, 0.3, 1.0, and 3.0 micrograms/h was infused intra-arterially, and effects of several receptor antagonists on motilin-induced contractions were examined. RESULTS The immunoreactive motilin concentration of venous effluent showed that motilin at doses of 0.1 and 0.3 microgram/h was within the physiological range. Each dose of motilin induced phasic contractions in the isolated stomach, and a dose-related increase in frequency was observed, but not their mean amplitude. Atropine, hexamethonium, ICS205-930, BRL43694, phentolamine, yohimbine, and propranolol significantly inhibited motilin-induced contractions. Naloxone, methysergide, and timolol did not affect the response of motilin. Prazosin significantly increased the mean amplitude of motilin-induced contractions. CONCLUSIONS Physiological dose of motilin can initiate phasic contractions in the stomach independently of the presence of the extrinsic nerves. The results suggest that cholinergic pathway, 5-hydroxytryptamine (HT)3 receptors, and alpha receptors are involved in the motilin-induced contractions.