The effects of motilin on gallbladder and sphincter of Oddi motor activity and transphincteric flow were studied in the cat in vivo. Motilin at doses of up to 1 microgram/kg had no effect on gallbladder motility. Increasing doses of intraarterial motilin (32-256 ng/kg) caused an increase in tonic pressures and in the force of phasic contractions of the sphincter of Oddi (p less than 0.001). Simultaneous measurements of myoelectric activity and transphincteric flow showed that motilin caused an increase in the frequency of spike bursts with a concomitant dose-dependent decrease in flow (p less than 0.001). The action of motilin was unaffected by extrinsic denervation (cervical vagotomy or splanchnicectomy), hexamethonium, and propranolol. Atropine, methysergide, or phentolamine partially blocked the excitatory effect of motilin (p less than 0.05), whereas tetrodotoxin, a combination of atropine and methysergide, or naloxone completely blocked its effects (p less than 0.001). It is concluded that (a) motilin decreases sphincter of Oddi flow by increasing its motor activity and (b) motilin activates an intramural excitatory pathway that appears to consist of opiate, serotonergic, and cholinergic neurons.