Free radicals are reportedly involved in mucosal injury, including NH4OH-induced gastric lesions, but the kind, location and origin of radical generation have yet to be clarified. We developed the non-invasive measurement of reactive oxygen species (ROS) in stomach, and applied to mucosal injury. NH4OH-induced gastric lesions were prepared in rats, which were then given a nitroxyl probe intragastrically or intravenously, and the spectra of the gastric region were obtained by in vivo 300 MHz electron spin resonance (ESR) spectroscopy. The spectral change of the nitroxyl probe administered intragastrically was significantly enhanced 30 min after NH4OH administration, but no change occurred when the probe was given by intravenous injection. The enhanced change was confirmed to be due to *OH generation, because it was completely suppressed by mannitol, catalase and desferrioxamine (DFO), and was not observed in neutropenic rats. NH4OH-induced neutrophil infiltration of the gastric mucosa was suppressed by intravenous injection of superoxide dismutase (SOD) or catalase, or by administration of allopurinol. The present study provided the direct evidence in NH4OH-treated living rats that *OH produced from O2*- derived from neutrophils caused gastric lesion formation, while O2*- or H2O2 derived from the xanthine oxidase system in endothelial cells was involved in neutrophil infiltration.