Pancreatic ganglia are innervated by neurons in the gut and are formed by precursor cells that migrate into the pancreas from the bowel. The innervation of the pancreas, therefore, may be considered an extension of the enteric nervous system. NADPH-diaphorase is present in a subset of enteric neurons. We investigated the presence of NADPH-diaphorase in the enteropancreatic innervation, the contribution of extrinsic nerves to the NADPH-diaphorase-containing fibers of the gut and pancreas, and the coincident expression of NADPH-diaphorase NADPH-diaphorase in intrinsic neurons of these organs with neuropeptides. The possible role of nitric oxide in the neural regulation of pancreatic secretion was studied in isolated pancreatic lobules. Neuronal perikarya with NADPH-diaphorase activity were found in both Dogiel type I and type II neurons of the myenteric plexus of the stomach and duodenum. All galanin (GAL)-immunoreactive neurons and a small subset of vasoactive intestinal polypeptide (VIP)- and neuropeptide Y (NPY)-immunoreactive neurons contained NADPH-diaphorase activity. NADPH-diaphorase activity was also found in a subset of VIP and NPY-immunoreactive pancreatic neurons. Retrograde tracing with FluoroGold established that NADPH-diaphorase-containing neurons in the bowel project to the pancreas. NADPH-diaphorase-containing fibers were also found to be provided to both organs by neurons in dorsal root ganglia. Secretion of amylase was evoked by L-arginine. This effect was prevented by N(G)-nitro-L-arginine (L-NNA), which also inhibited VIP-stimulated secretion of amylase; however, L-NNA had no effect on amylase secretion stimulated by carbachol. These results provide support for the hypothesis that nitric oxide plays a role in the neural regulation of pancreatic secretion.