The sphincter of Oddi has a typical nonadrenergic noncholinergic inhibitory innervation; however, the transmitter of this inhibition has not been identified. The aim of the present study was to evaluate whether metabolites of the L-arginine – nitric oxide synthase pathway mediate neural inhibition in the sphincter of Oddi of the opossum. Electrical field stimulation at various frequencies (3, 5, and 10 pulses/s), performed in the presence of guanethidine (10−6 M) to exclude adrenergic responses, caused a slight, but significant excitatory response of the sphincter of Oddi. The responses were more pronounced at the duodenal side of the sphincter of Oddi than on the hepatic side. When the electrical field stimulation was repeated after blockading muscarinic receptors, using atropine (10−6 M), a potent inhibitory response was obtained. The inhibitory response to each of the various stimulation parameters was similar. Addition of L-arginine methyl ester (L-NAME, 2 × 10−4 M) abolished and reversed the inhibitory effect of electrical field stimulation, resulting in a potent stimulatory effect. Higher frequencies (5 and 10 pulses/s) were more potent in causing a stimulatory response than lower frequencies (3 pulses/s). The excitatory effect of electrical field stimulation was blocked or reversed to inhibition when the amino acid L-arginine (2 × 10−3 M) was added to the bath. In a second series of experiments, the inhibitory effect of electrical field stimulation in the presence of atropine and guanethidine was not prevented after the addition of methylene blue (5 × 10−5 M), a substance that, in vascular smooth muscle, has been demonstrated to block cyclic GMP dependent inhibitory responses. These data demonstrate that the sphincter of Oddi is characterized by an excitatory innervation that is partly cholinergic and partly nonadrenergic noncholinergic (NANC), while the NANC inhibitory response of this sphincter muscle is mediated by the release of endogenous nitric oxide or related compounds.Key words: nonadrenergic, noncholinergic, nitric oxide, L-arginine, sphincter of Oddi, methylene blue.