HCO3- exit across the basolateral membrane of the kidney proximal tubule cell is mediated via an electrogenic Na+:HCO3- cotransporter. In these experiments, we have studied the effect of internal pH on the activity of the Na+:HCO3- cotransport system in basolateral membrane vesicles isolated from rabbit renal cortex. Equilibrium thermodynamics predicts that in the presence of constant intravesicular concentration of Na+, an increasing concentration of HCO3- will be associated with an increasing driving force for Na+:HCO3- cotransport across the vesicles. Our experimental approach was to preequilibrate the membrane vesicles with 1 mM 22Na+ at pHi 6.8-8.0 and known concentrations of HCO3-. The vesicles were diluted 1:100 into Na(+)-free solution at pH 7.4 and the net flux of 22Na+ was assayed over 5 s. The results demonstrate that the net flux of Na+ was significantly higher at pHi 7.2 than pHi 8.0 despite much higher [HCO3-] at pHi 8.0. This suggests that an internal pH-sensitive site regulates the activity of the Na+:HCO3- cotransporter. This modifier site inhibits the cotransporter at alkaline pH despite significant base concentration and is maximally functional around physiologic pH. The combination of modifier sites on the luminal Na+/H+ exchanger and the basolateral Na+:HCO3- cotransporter should help maintain intracellular pH in a narrow range with changes in extracellular pH.