A dual-excitation inverted confocal laser-scanning microscope has been developed for measuring intracellular pH (pHi) using 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) in individual cells in the isolated perfused cortical collecting tubule (CCT). This new microscope has superior depth discrimination, which eliminates the contribution of fluorescence information from cells outside the plane of focus. pHi was monitored in real time from a spot 0.55 microns in diameter within a single cell. Experiments were performed to examine the apical and basolateral membrane H+/base transport properties of single principal cells. The results indicate that principal cells possess a basolateral membrane Na(+)-independent Cl-/base exchanger, a Na(+)-H+ antiporter, and a Na+/base cotransporter. No evidence was found for an apical membrane Na(+)-independent Cl-/base exchanger. The data provide evidence for base efflux pathways in the principal cell and are compatible with the hypothesis that principal cells contribute importantly to H+/base transport in the CCT. The new methodology described in this report can be applied to other epithelia that are optically heterogeneous in the depth dimension.