Cytosolic pH (pHi) is a critically regulated determinant of intracellular function. Several mechanisms for pHi regulation in different tissues have been found, such as direct proton pumping, Na/H exchange, Cl/HCO3 exchange, NaHCO3 cotransport, and Na/H/Cl/HCO3 obligatorily linked. All these studies have used either single cells or cell populations assumed to be behaving homogeneously. Most tissues consist of more than one cell type, so it would be desirable to examine pHi regulation simultaneously in many identified individual cells, particularly in epithelia where disaggregation and purification of isolated cells destroys the normal distinction between luminal and serosal environments. We have used a pH-sensitive fluorescent dye, BCECF (2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein) and digital image processing to study pHi regulation simultaneously in the oxyntic cells (OC) and chief cells (CC) of gastric glands isolated from rabbit stomach. CCs become markedly more acidic upon removal of external Na (Nao), but pHi is restored rapidly on return to normal Nao, with or without Cl. Oxyntic cell pHi is much less affected by Nao. Conversely, OCs become strongly more alkaline on removal of external Cl (Clo), pHi being restored when Clo is replaced with or without Na, whereas CCs are relatively insensitive to Clo. Therefore, Na/H exchange is dominant over Cl/HCO3 exchange in CCs, but in the neighbouring OCs, Cl/HCO3 outweighs the Na/H mechanism, a heterogeneity that correlates with the functions of the two cell types.