The mechanism that regulates the synthesis of nitric oxide synthase (NOS), a key enzyme responsible for NO production in the myenteric plexus, remains unknown. We investigated the roles of the vagal nerve and nicotinic synapses in the mediation of NOS synthesis in the gastric myenteric plexus in rats. Truncal vagotomy and administration of hexamethonium significantly reduced nonadrenergic, noncholinergic relaxation, the catalytic activity of NOS, the number of NOS-immunoreactive cells, and the density of NOS-immunoreactive bands and NOS mRNA bands obtained from gastric tissue. These results suggest that NOS expression in the gastric myenteric plexus is controlled by the vagal nerve and nicotinic synapses. We also investigated if stimulation of the nicotinic receptor increases neuronal NOS (nNOS) expression in cultured gastric myenteric ganglia. Incubation of cultured gastric myenteric ganglia with the nicotinic receptor agonist, 1,1-dimethyl-4-phenylpiperizinium (DMPP, 10(-10)-10(-7) M), for 24 h significantly increased the number of nNOS-immunoreactive cells and the density of immunoreactive nNOS bands and nNOS mRNA bands. nNOS mRNA expression stimulated by DMPP was antagonized by a protein kinase C antagonist, a phospholipase C inhibitor, and an intracellular Ca2+ chelator. We concluded that activation of the nicotinic receptor stimulates a Ca2+-dependent protein kinase C pathway, which in turn, upregulates nNOS mRNA expression and nNOS synthesis in the gastric myenteric plexus.