A secretin-releasing activity exists in the upper small intestine and pancreatic juice in the rat and the dog. Group I pancreatic phospholipase A₂ (PLA₂) in canine pancreatic juice and porcine pancreatic PLA₂ stimulate the release of secretin from both STC-1 cells and a secretin-producing cell (S cell)-enriched preparation isolated from rat duodenal mucosa. We investigated the distribution and release of pancreatic PLA₂-like immunoreactivity in the gastrointestinal tract and the role of PLA₂ on the release of secretin and pancreatic exocrine secretion in response to duodenal acidification in anesthetized rats. PLA₂-like immunoreactivity was detected in the mucosa throughout the gastrointestinal tract. High concentrations of PLA₂ were found in both the small intestine and the pancreas. Duodenal acidification significantly increased the release of PLA₂ from the upper small intestine (385% over basal secretion). Intravenous infusion of an anti-PLA₂ serum (anti-PLA₂) dose-dependently inhibited the release of secretin and pancreatic exocrine secretion in response to duodenal acid perfusion. Preincubation of the concentrate of intestinal acid perfusate (10-fold) from donor rats with the anti-PLA₂ significantly suppressed its stimulation of secretin release and pancreatic exocrine secretion in recipient rats. We conclude that pancreatic PLA₂ also functions as a secretin-releasing factor in the small intestine that mediates acid-stimulated release of secretin in rats.