In the stomach, production of prostaglandins by cyclo-oxygenase (COX) is believed to be important in mucosal defence. We tested the hypothesis that endogenous COX activity is required for protective gastric surface pH control. Intact stomachs of anaesthetized mice were perfused with a weakly buffered solution (150 mM NaCl + 4 mM Homopipes) at pH values from 2.5 to 7.0. Gastric effluents were collected to measure pH and estimate amounts of acid or alkali secretion in nanomoles secreted per minute. A switch from net acid to net alkali secretion was seen in response to acidifying luminal pH with an apparent 'set point' between pH 4 and 5. At luminal pH 3, the net alkali secretion (12.7 ± 2.8 nmol OH^- equivalents min^-1) was abolished (2.2 ± 1.7 nmol OH^- min^-1) by the non-specific COX inhibitor indomethacin (5 mg kg^-1 I.P.). Similar inhibition was observed using a COX-1 inhibitor (SC-560; 10 mg kg^-1 I.P.), but not a COX-2 inhibitor (NS-398; 10 mg kg^-1 I.P.). Subsequent treatment with 16,16-dimethyl prostaglandin E₂ (dm-PGE₂; 1 mg kg^-1 I.P.) rescued the alkali secretion (21.8 ± 2.7 nmol OH^- min^-1). In either the absence or presence of the H⁺,K⁺-ATPase inhibitor omeprazole (60 mg kg^-1 I.P.), indomethacin blocked similar amounts of net alkali secretion (10.5 ± 2.7 and 16.4 ± 3.4 nmol OH^- min^-1, respectively). We also used in vivo confocal microscopy to examine pH near the mucosal surface. The gastric mucosal surface of anaesthetized mice was exposed and mucosal surface pH was imaged using the fluorescence intensity ratio of Cl-NERF as a pH indicator. Results showed a switch from a continuous net acid to net alkali secretion by the stomach in response to changing superfusate pH from 5 to 3. At luminal pH 3, the relatively alkaline surface pH (4.3 ± 0.1) was acidified (3.6 ± 0.2) by indomethacin, and subsequent dm-PGE₂ restored surface pH (4.2 ± 0.2). We conclude that the pre-epithelial alkaline layer is regulated by endogenous COX activity.