Cycloxygenase-1 (Cox-1) contributes to gastric defense of healthy tissue, but the role in protection of gastric epithelium after minor, acute damage has been difficult to study in vivo. Using two-photon light absorption to create microscopic gastric damage in anesthetized mice, the acute response of surface cells to injury could be monitored using in vivo microscopy within seconds after injury. Using exogenous (Cl-NERF) and endogenous fluorophores, extracellular pH and cell death were monitored in real-time during the damage and repair cycle. Two-photon damage was initiated by scanning ~200 µm² of gastric surface cells with high laser intensity, causing rapid bleaching of NAD(P)H fluorescence in optically targeted cells. In both Cox-1 +/- and -/- mice, a similar initial damage area expanded to include bystander epithelial cells over the next 2-5 min, with larger maximal damage noted in Cox-1 -/- mice. The maximal damage size seen in Cox-1 -/- mice could be reduced by exogenous dimethyl-PGE₂. All damaged cells exfoliated and the underlying epithelium was coincidently repaired over a time interval that was briefer in Cox-1 +/- (12±2 min, n=12) than in Cox-1 -/- (24±4 min, n=14). Directly after damage, pH increased transiently in the juxtamucosal layer (maximal at 3-6 min). Smaller peak pH change was noted in Cox-1 -/- (pH=0.3±0.04) than in +/- mice (pH=0.6±0.2). Recovery to normal surface pH took longer in Cox-1 -/- (27±5 min) than in +/- mice (12±1 min). In conclusion, constitutive loss of Cox-1 leaves the gastric mucosa more prone to damage and slowed repair of micro-lesions.