The second phase of pancreatic polypeptide (PP) release after a meal is thought to occur via a vagal-cholinergic-dependent pathway acting in concert with a putative humoral factor of gut origin. To identify this enteric factor(s), extracts of canine duodenal mucosa were evaluated for PP secretagogues using canine pseudoislets in a column-perifusion bioassay system. Two thermostable agents with PP secretagogue activity have been found. The smaller and predominant peptide had an apparent molecular weight slightly larger than insulin and migrated on Sephadex G-50 columns at a distribution coefficient of 0.47 +/- 0.01. This secretagogue consistently elicited 1.3-1.5 times greater PP secretory response when compared to the larger peptide, which migrated at an average distribution coefficient of 0.29 +/- 0.02. Chromatographically similar activities were found in extracts of duodenal venous plasma collected 2 h after a meal. These PP secretagogues are presumed to be unique to the duodenal mucosa since they were not observed in extracts of liver or heart. These results show that the duodenal mucosa contains PP secretagogues larger than those previously demonstrated and further support the notion that a substance of gut origin participates in the mechanism of postprandial PP release.