The intracellular pH (pHi) of isolated hamster intestinal cells was determined with three techniques: the null-point method with digitonin, the distribution of the weak acid 5,5-dimethyloxazolidine-2,4-dione, and the trapped fluorescent indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In physiologic saline, the pHi determined with the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein was 7.00 +/- 0.03 (n = 20) but was 6.68 +/- 0.04 (n = 25) when there was no Na+ in the medium, indicating an important role of external Na+ in maintaining pHi at neutral level. When Na+ was added to an incubation medium lacking the ion, the pHi increased. The time-course of this alkalinization depended on the Na+ concentration, whereas K+ had only a slight effect. Amiloride (1 mM) completely inhibited the Na+ effect, thereby showing the basic role of the Na+/H+ antiport in the regulation of pHi. The effect of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and furosemide showed that there also exists an anionic component in the process, probably of the Cl-/OH- (HCO3-) exchange type. These results show that the amiloride-sensitive Na+/H+ antiport plays an important but not exclusive role in maintaining the pHi neutral or slightly alkaline in isolated intestinal cells from the hamster.