In conscious dogs with gastric fistula and platinum electrodes on the antrum, duodenum and jejunum, IV atropine 100 micrograms/kg/hr and hexamethonium 10 mg/kg/hr, blocked cyclic increases in fasting plasma motilin concentration (PMC) and spontaneous migrating myoelectric complexes (MMCs) of both antrum and duodenum. The two drugs also blocked occurrence of premature MMCs produced by synthetic porcine motilin. In anesthetized dogs, electrical stimulation of cervical vagi with stimulation parameters: 9 V, 10 c/s, 5 msec, caused a significant increase in both portal and femoral venous PMC which was blocked by atropine. Fractionations of vagus nerve extracts by gel filtration using Sephadex G-50 superfine column revealed most of motilin-like immunoreactivity (MLI) with the same mobility as pure porcine motilin. Studies suggest that cholinergic influence plays a significant role on release of motilin.