Antropyloroduodenal motility was recorded in seven anesthetized dogs to assess the role of nitric oxide and L-arginine metabolites in nonadrenergic noncholinergic (NANC) mediation of pyloric relaxation. Pyloric activity induced by duodenal field stimulation was inhibited by antral field stimulation and electrical vagal stimulation. Intra-arterial NG-L-arginine-methyl-ester (L-NAME) reduced the inhibition from antral or vagal stimulation (P less than 0.05). Intravenous infusion of L-NAME also blocked the inhibitory effect of vagal and antral stimulation but left the tetrodotoxin-insensitive action of intra-arterial vasoactive intestinal peptide (VIP) and sodium nitroprusside unchanged. L-Arginine reversed the effect of L-NAME whereas D-arginine did not. L-NAME enhanced pyloric contractions to intra-arterial acetylcholine. The NANC inhibition of the substance P-stimulated pyloric response in vitro was blocked by L-NAME and reversed by addition of L-arginine. Sodium nitroprusside was effective as a relaxant in vitro but VIP was not. These data suggest that metabolites of L-arginine mediate neural inhibition of canine pyloric motor activity.