The present experiments were performed to determine pathways responsible for arachidonic acid release stimulated by cholecystokinin (CCK) and phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA), and the roles of pathways in the secretory response in dispersed acini from guinea pig pancreas. Both CCK-octapeptide (CCK-OP) and PMA increased intracellular arachidonic acid. To determine the source of released arachidonic acid, we measured the effects of PMA and CCK-OP on cellular 1,2-diacylglycerol and lysophosphatidylcholine (LPC) and of diglyceride lipase inhibitor RHC 80267 on [3H]arachidonic acid release. Both PMA and CCK-OP increased 1,2-diacylglycerol and LPC. RHC 80267 had no effect on LPC but inhibited the increase in [3H]arachidonic acid release with a concentration of CCK-OP that was maximal for enzyme secretion. The increase in [3H]arachidonic acid release with PMA or a supramaximal concentration of CCK-OP was not inhibited by RHC 80267. In parallel fashion, RHC 80267 inhibited amylase release caused by maximally effective concentrations of CCK-OP but not that caused by PMA or by supramaximally effective concentrations of CCK-OP. Arachidonic acid stimulated amylase release. Exogenous addition of phospholipase A2 caused increases in [3H]arachidonic acid release, LPC formation, and amylase release. The results indicate that there are at least two pathways responsible for the increase in free cellular arachidonic acid stimulated by pancreatic agonists. One is sequential action of phospholipase C and diglyceride lipase on phosphatidylinositol. The other is a phospholipase A action on phosphatidylcholine. The results also suggest a stimulatory role for both pathways in the secretory response.