In gastric mucosal injury, nitric oxide (NO) plays both cytoprotective and cytotoxic roles, and the NO level is one determinant of these dual roles. We employed electron paramagnetic resonance (EPR)-spectrometry combined with an NO-trapping technique to directly evaluate NO production in ethanol-induced gastric injury in rats. The rat stomach, mounted on an ex vivo chamber, was perfused with ethanol (12.5 and 43%), and NO levels in mucosal tissues were measured during perfusion. Luminal nitrite/nitrate (NOx) content, mucosal blood flow, area of mucosal injury, transmucosal potential difference (PD), and luminal pH were simultaneously monitored with/without preadministration of the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME). NO levels in the gastric tissue increased during ethanol perfusion, and luminal NOx levels increased after the perfusion, accompanying an increase in the area of mucosal injury and changes in physiological parameters. Preadministration of L-NAME aggravated the gastric mucosal damage and suppressed increases in mucosal blood flow in a dose-dependent manner. These results demonstrate that endogenous NO produced in ethanol-induced gastric injury contributes to maintenance of mucosal integrity via regulation of mucosal blood flow.