1. Single-barrelled pH-sensitive microelectrodes filled with liquid ion exchanger were used to study the layer of microclimate pH in the vicinity of the surface of rat jejunum in vitro. 2. During perfusion with a Na+-containing solution of pH 7.30, a layer having a pH gradient ranging from 7.30 (pH of the luminal bulk phase) to 6.05 +/- 0.03 (pH of the deepest region) was detected in eighteen different animals. The thickness of the layer was estimated to be 600-700 microns. No regional difference was seen along the height of the villus. 3. The addition of D-glucose to the perfusion solution significantly augmented the acidity of the deepest region without changing the thickness of the layer. On the other hand, the elimination of Na+ from the perfusion solution caused a significant reduction of the pH gradient. The lowest pH changed from a control value of 6.18 +/- 0.15 (n = 13 measurements from three animals) to 6.46 +/- 0.06 (n = 13). The gradient was sensitive to amiloride in the presence of Na+, K+, Ca2+ and Cl- had no significant effect on the microclimate pH. 4. Depletion of the surface mucus by treatment with dithiothreitol significantly raised the pH of the deepest region. 5. Glycylglycine and L-carnosine were found to reduce the microclimate pH gradient significantly, while glycine did not. 6. These results indicate that H+ secretion by the Na+-H+ antiport and the formation of mucus layer are important factors for maintaining the microclimate pH layer, and that H+-coupled co-transport, such as H+-dipeptide co-transport, causes a significant diminution of the microclimate pH gradient.