Recent studies suggested the involvement of arachidonic acid in the mediation of pancreatic amylase release. However, an effect of carbamylcholine on arachidonic acid release has not yet been reported in the exocrine pancreas. This study was performed to evaluate the effect of carbamylcholine on arachidonic acid release and determine the underlying intracellular mechanisms. From enzymatic assays, phospholipase A(2) and diacylglycerol lipase were activated by carbamylcholine and these activations were inhibited by the phospholipase A(2) inhibitors, mepacrine and aristolochic acid, and by the diacylglycerol lipase inhibitor RHC 80267. Carbamylcholine also increased arachidonic acid release in a concentration-dependent manner. Both phospholipase A(2) and diacylglycerol inhibitors partially inhibited carbamylcholine-stimulated arachidonic acid release. The phospholipase C inhibitor U73122 and the protein kinase C inhibitor staurosporine also caused partial inhibition. Arachidonic acid release by carbamylcholine was suppressed by the simultaneous addition of RHC 80267 with either phospholipase A(2) inhibitors. Our data demonstrate that phospholipase A(2) and diacylglycerol lipase are activated and arachidonic acid is released in pancreatic acini by carbamylcholine. Dual pathways are responsible for carbamylcholine-induced arachidonic acid release. One such pathway involves the sequential action of phospholipase C, protein kinase C and diacylglycerol lipase, whereas the other involves phospholipase A(2) activation.