Intracellular pH (pHi) of the colonic tumor cell line HT29 cl. 19A was studied by microspectrofluorometry using the pH-sensitive dye BCECF. Single cells within a confluent monolayer grown in a polarized manner on permeable supports were examined. An amiloride-sensitive Na(+)-H+ exchange and a stilbene-insensitive Cl(-)-HCO3- exchange mechanism have been identified in the basolateral membrane. This Na(+)-H+ exchange mechanism in the basolateral membrane is an acid extruder, whereas the Cl(-)-HCO3- exchanger is an acid loader. Both of these in the basolateral membrane located mechanisms are important for the maintenance of intracellular pH in HT29 cl. 19A cells.