Here we show that the relaxation induced by stimulation of the vagus nerve in the presence of cholinergic (muscarinic) and adrenergic blockade in the isolated stomach of the guinea pig is mediated by nitric oxide (NO). This is substantiated by inhibition of vagal relaxation by NGmonomethyl-L-arginine, an inhibitor of NO synthesis. The effect of NG-monomethyl-L-arginine was partially reversed by coincubation with L-anine but not with D-arginne. NO activates soluble guanylate cyclase, and relaxation of the stomach induced by vagal stimulation was prevented by an inhibitor of soluble guanylate cyclase, methylene blue, further supporting our conclusions. The relaxant effect of vagal stimulation was also ablated by hexamethonium, an inhibitor of ganglionic nicotinic receptors, thereby showing that gangionic transmission did not rely on NO, through its release from pr onic neurons. However, hexamethonium did not inhibit the gastric relaxation brought about by increasing the inragastric pressure, which is also mediated by NO as previously described by us. The selective inhibition by hexamethonum of only the vagafly mediated relaxation but not of the pressure-induced relaxation of the stomach indicates the existence of at least two separate neuronal pathways able to generate NO and bring about gastric accommodation of food or fluid.Endothelium-derived relaxing factor, or nitric oxide (NO; ref. 1), plays important roles in the cardiovascular and immune systems (2, 3). Demonstration of the biosynthesis of NO by neuronal cells in the central nervous system (4) suggested a role for NO in intercellular communication in the brain. NO synthase, an NADPH-dependent dioxygenase that catalyzes formation of NO from one of the guanidino nitrogens of L-arginine, has been purified from the rat brain (5), sequenced, and expressed from its cDNA (6). The enzyme has been immunolocalized in nerve fibers and cell bodies of neurons in the brain and in the myenteric plexus in the gastrointestinal tract (7). Moreover, the myenteric plexus is an abundant source of neurons showing NADPH diaphorase activity (8), and NADPH diaphorase is a NO synthase (9). All these findings suggest the existence of nerves that release NO as a transmitter ("nitroxergic" nerves) in the central and peripheral nervous system. Inhibitory nerves were described in the stomach (10) and nonadrenergic, noncholinergic (NANC) inhibitory nerves have now been recognized in many parts of the gastrointestinal tract (11). Recent evidence demonstrates that these nerves are nitroxergic at some sites. Thus, the generation and release of NO has been demonstrated on electrical stimulation of NANC nerves in the canine ileocolonic junction (12) and rat stomach fundus (13). Also, the inhibition of NO formation prevents the relaxation of gastrointestinal smooth muscle, induced by electrical field stimulation of NANC nerves (13-15) or by other putative NANC neurotransmitters such as vasoactive intestinal peptide (VIP) (15), adenosine 5'-triphosphate (ATP), or -t-aminobutyric acid (1...