This study was designed to determine the pH of the intestinal surface in the rat jejunum in an attempt to investigate the effect of potential damaging agents on the microclimate-pH. A significant pH gradient was observed between the mucosal surface and the bulk phase; however, the microclimate-pH usually ranged from 6.5 to 7.5, irrespective of the wide range in the bulk pH. When the bulk pH was either 7.3 or 4, the microclimate-pH was approximately 6.7, while the microclimate-pH was approximately 7.4 when the bulk pH was raised to 9. The distance of the pH gradient was found to be approximately 900-1300 microns. After treatment with ouabain (10 mM) or amiloride (8 mM), or replacement of Na+ with Li+, the microclimate-pH significantly increased compared with the control (buffer solution). Chlorpromazine (2 mM) and ouabain also significantly increased the microclimate-pH, but at the same time, chlorpromazine decreased the bulk pH so that a more marked pH gradient was observed. Both aspirin (10 mM) and acetazolamide (80 mg/kg, iv) significantly decreased the microclimate-pH under the condition of the acidic bulk phase pH. N-Acetyl-L-cysteine (5%) induced marked changes in microclimate-pH, the direction depending upon the bulk phase pH. Neither taurocholic acid (10 mM) nor glucose (10 mM) significantly changed the microclimate pH.