BACKGROUND Nitric oxide acts as a neurotransmitter of nonadrenergic, noncholinergic pathways. The present study examined the role of the L-arginine/NO pathway on sphincter of Oddi motility. METHODS In anesthetized guinea pigs and rabbits, intracholedochal pressure was recorded by a perfused catheter whose open tip was maintained close to the sphincter of Oddi lumen. The contractile responses to cholecystokinin or bethanechol were recorded before and after treatment with either a specific NO synthetase inhibitor (NG-nitro-L-arginine methyl ester) or a donor of NO (sodium nitroprusside). The effect of NO synthase inhibition on isometric tension generated by muscle from rabbit sphincter of Oddi was tested in vitro. RESULTS Tonic pressure and phasic contractions significantly increased after NO synthase inhibition; responses to cholecystokinin and bethanechol were enhanced. In contrast, sodium nitroprusside reduced the response to cholecystokinin. Isolated muscle from rabbit sphincter of Oddi relaxed in response to the nicotinic agonist dimethyl-4-phenylpiperazinium. Relaxation was eliminated by either the neurotoxin tetrodotoxin or by NG-nitro-L-arginine methyl ester. NO synthase inhibition also suppressed the relaxatory response induced by electrical field stimulation. Calcium-dependent activity of NO synthase was detected in fresh homogenates from guinea pig and rabbit sphincter of Oddi tissue. CONCLUSIONS NO that is locally generated by a constitutive NO synthase regulates sphincter of Oddi motor function.