Rabbit esophageal epithelium grown in primary culture enabled the study of intracellular pH (pHi) regulation at two distinct stages in the life cycle of the epithelial cell: basal and mature squamous. pHi was measured in single basal and mature squamous cells after loading with the fluorescent probe 2',7'-bis(carboxyethyl)-5(and -6)carboxyfluorescein at 25 degrees C in a nominally bicarbonate-free HEPES buffer. The results revealed that the resting pHi was higher and the intrinsic buffer capacity lower (at pH values less than or equal to 7.6) in basal compared with mature squamous cells. In addition, both types recovered from an acid load (NH4Cl prepulse) by an Na(+)-dependent, amiloride-inhibitable process consistent with an Na+/H+ antiporter. However, hydrogen ion extrusion rates by the Na+/H+ antiporter, even after taking into account buffer capacity and acid loading, were two to four times as fast for basal as for mature squamous cells. Further, mature squamous cell but not basal cell recovery from an acid load deteriorated with time (3 weeks) in culture. These results establish the use of primary cultures for studying pHi regulation at different stages in the epithelial cell life cycle and document that basal and mature squamous cells show Na+/H+ antiport activity for extrusion of an acid load but that this activity diminishes in effectiveness as the cell matures.