This study provides mechanical and electrophysiological evidence to show that a metabolite of arginine, not vasoactive intestinal peptide (VIP), is the putative nonadrenergic noncholinergic (NANC) inhibitory mediator in canine and opossum lower esophageal sphincters (LES). Relaxations of spontaneous active tension by electrical field stimulation (FS) at parameters that induced tetrodotoxin (TTX)-sensitive responses were abolished by L-Nω-arginine methyl ester (L-NAME) at 10−4 M and restored by L-arginine (10−3 M) but not D-arginine (10−3 M). TTX-insensitive relaxations to 5-ms pulses were unaffected by L-NAME, L- or D-arginine. VIP (10−6 M) caused maximum relaxations of basal tension in both the opossum and canine LES. However these relaxations, unlike those from FS were unaffected by L-NAME. Methylene blue (5 × 10−5 M) increased basal tension of the LES in each species, and did not inhibit the relaxation to FS or VIP, but often increased the amplitudes of these responses due to the increase in basal tension. In parallel experiments NANC inhibition of body circular muscle from opossum esophagus was abolished by methylene blue. Electrophysiological studies using micro-electrodes revealed that NANC inhibition was associated with inhibitory junction potentials in the canine LES. These were inhibited by L-NAME and restored by L-arginine but not D-arginine. In contrast, 10−6 M VIP in canine LES did not induce any change in membrane potential during a 20-min superfusion. Sodium nitroprusside also hyperpolarized sphincteric muscle and its effects were not affected by L-NAME. We conclude that esophageal sphincter NANC nerve initiation of inhibitory junction potentials or relaxations depend on a mediator produced from L-arginine, like those of esophageal body circular muscle. This mediator resembles sodium nitroprusside in its actions. However, either the NANC mediator or the response to it differs in body circular muscle and LES since only the former was clearly sensitive to methylene blue. Moreover, TTX-insensitive relaxations to long pulse durations were not mediated by nitric oxide-related mechanisms because L-NAME had no effect on them.Key words: nitric oxide, vasoactive intestinal peptide, sphincter relaxation, inhibitory junction potentials.