Using a single sucrose gap apparatus, experiments were performed to determine the involvement of nitric oxide (NO) in the generation of nonadrenergic, noncholinergic (NANC) inhibitory junction potentials in circular muscle of rat proximal colon. Inhibitors of NO synthase, N omega-nitro-L-arginine and its methyl ester, reduced the amplitude of the electrically evoked inhibitory junction potentials, without affecting membrane resting potential. Such an effect was stereospecific and it was prevented by L-arginine but not by D-arginine. Sodium nitroprusside induced a tetrodotoxin-resistant hyperpolarization, which was not affected by NO synthase inhibitors. Apamin reduced sodium nitroprusside induced hyperpolarization, as well as NANC inhibitory junction potentials, and alpha-chymotrypsin decreased the amplitude of electrical field stimulation evoked responses. Residual responses after NO synthase inhibitors or after alpha-chymotrypsin were further reduced by pretreatment with alpha-chymotrypsin or NO synthase inhibitors, respectively. These results suggest that, in rat colonic circular muscle, NO plays an important role in NANC inhibitory junction potential generation. However, another mechanism, peptidergic in nature, is also involved.