BACKGROUND: The authors previously reported that ketamine and midazolam inhibited nitric oxide-mediated nonadrenergic noncholinergic (NANC) lower esophageal sphincter (LES) relaxation nitric oxide-3',5'-cyclic guanosine monophosphate pathway modulation. The mechanisms inhibiting the NANC relaxation by ketamine and midazolam were investigated. METHODS: The isometric tension of circular distal esophageal muscle strips from Japanese White rabbits was examined. NANC relaxation was induced by KCl (30 mm) in the presence of atropine (3 x 10(-6) m) and guanethidine (3 x 10(-6) m). Nitric oxide synthase activity in the absence and presence of ketamine and midazolam was analyzed using the biochemical conversion of L-[3H]arginine to L-[3H]citrulline. RESULTS: The ketamine-induced inhibition of the NANC relaxation was partly reversed by superoxide dismutase (200, 400 U/ml) but not by catalase (100 U/ml). Ketamine concentration-dependently inhibited the relaxation induced by N-ethylethanamine:1,1-diethyl-2-hydroxy-2-nitrosohydrazine (diethylamine NONOate) and S-nitrosoglutathione. The NANC relaxation itself was not affected by superoxide dismutase. The midazolam-induced inhibition of the NANC relaxation was reversed neither by superoxide dismutase nor by catalase, and midazolam did not affect the relaxations induced by nitric oxide donors. The nitric oxide synthase activity was concentration-dependently suppressed by midazolam, but there was no marked effect of ketamine. Pyrogallol, a superoxide generator, inhibited the NANC and the diethylamine NONOate-induced relaxations. The pyrogallol-induced inhibition of the NANC relaxation was reversed by superoxide dismutase. CONCLUSION: These findings suggest that ketamine inhibits NANC LES relaxation by the extracellular production of superoxide anion, and that midazolam inhibits it by the inhibition of nitric oxide synthase activity.