Activation of intrinsic nonadrenergic noncholinergic (NANC) esophageal nerves during peristalsis or by electrical field stimulation (EFS) in vitro produces a hyperpolarization followed by a depolarization of the circular smooth muscle of the opossum esophagus. N omega-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide synthase, and nitric oxide (NO) were used to test the hypothesis that NO or a NO-containing compound is a mediator of this NANC nerve-induced hyperpolarization of circular esophageal smooth muscle. The transmembrane potential difference of esophageal circular smooth muscle cells was recorded with glass microelectrodes. Nerve-mediated membrane responses were evoked by single electrical pulses of 0.5 ms duration and 50 V amplitude. L-NNA abolished the initial hyperpolarization and reduced the amplitude of and the time to maximal depolarization. L-Arginine (1 mM), the substrate for NO synthase, antagonized the effect of L-NNA. Exogenous NO produced hyperpolarization of the smooth muscle membrane potential and attenuated the amplitudes of EFS-induced hyperpolarization and depolarization. The effect of NO was blocked neither by L-NNA nor by tetrodotoxin (1 microM). The data support the hypothesis that NO or a NO-containing compound mediates NANC nerve-induced responses of the esophageal smooth muscle membrane.