The presence of an unequilibrated region adjacent to the mucosal surface of the small intestine has long been suggested. Controversy remains as to the characteristics of this region. This study was designed to further define these characteristics. Our studies demonstrated by two independent methods, pH electrode measurement and dye indicator, that a low-pH compartment does exist at the surface of the intestine. The thickness of this microclimate was estimated to be 700 microns, comparable with that described for the unstirred water layer. Maintenance of the low-pH compartment is due to the presence of a mucus coating rather than hydrogen ion secretion. We postulate that the mucus functions as an ampholyte and restricts hydrogen ion movement in its matrix. The acidic microclimate, unstirred water layer, and the mucus coating might in fact represent a common phenomenon.