The mechanism of associated fluctuations in plasma motilin and pancreatic polypeptide (PP) concentrations was studied in fasted conscious dogs while gastric motility was monitored. Plasma motilin and PP concentrations were measured by radioimmunoassay. In intact normal dogs, exogenous motilin (0.03-0.3 g/kg) stimulated dose-related release of PP, but PP did not stimulate motilin release. Motilin-induced PP release was completely inhibited by pretreatment with cholinergic blockers and a 5-hydroxytryptamine3 (5-HT3) receptor antagonist, and by vagotomy. The cyclic release of PP was abolished after vagotomy and duodenectomy. However, PP release stimulated by exogenous motilin was apparent after duodenectomy but not after vagotomy. In conclusion, motilin appears to stimulate PP release via vagal, cholinergic muscarinic pathways involving 5-HT3 receptors and to act as a biosignal controlling PP release by mediating the interdigestive periodic changes in the duodenum to the center of the autonomic nervous system. This represents a new role for motilin.