1. The mechanisms for regulating the intracellular pH (pHi) level during oligopeptide absorption were investigated in the enterocytes from guinea-pig ileum by identifying the acid-base transporters responsible for extruding H+ that enters the cell through the H+-oligopeptide cotransporter. 2. The pHi level was measured by microfluorometry in an isolated villus tip loaded with the pH-sensitive fluoroprobe 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The oligopeptide-induced increment in the short-circuit current (Isc) was determined in a mucosal sheet in Ussing chambers. A CO2/HCO3--buffered solution was used. 3. The superfusion of glycylglycine (Gly-Gly, l0 mM) caused a decrease in pHi level, which returned to the basal level after removing Gly-Gly. This pHi recovery was strongly dependent on extracellular Na+. Amiloride partially inhibited the pHi recovery rate with an IC50 value of 41 microM, the maximum inhibition being approximately 70%. In the presence of amiloride at its maximum concentration (0.3 mM), the addition of 0.6 mM DIDS caused a further decrease, but did not abolish the pHi recovery rate. In the absence of CO2 and HCO3-, the pHi recovery was almost completely abolished by 0.3 mM amiloride. 4. The intracellular H+ accumulation induced by 0.3 mM amiloride or by 0.6 mM DIDS, as estimated from the pHi decrease and buffer capacity, was significantly greater during Gly-Gly superfusion than under resting conditions. 5. The increase in Isc induced by luminal glycylproline was attenuated by either removing serosal Na+ or by adding 0.5 mM amiloride or 0.6 mM DIDS to the serosal side. 6. We conclude that both Na+-dependent, amiloride-sensitive acid extrusion, probably by the Na+-H+ exchanger, and Na+- and HCO3--dependent, DIDS-sensitive acid extrusion, possibly by the Na+-HCO3- cotransporter, are involved in extruding H+ that enters cells by the H+-oligopeptide cotransport. It is proposed that these acid extrusion (or base loading) mechanisms are present in the basolateral membrane and are important for maintaining oligopeptide absorption, as well as the acid extrusion mechanism in the apical membrane.