Heterogeneity of cytosolic pH was studied with compounds that distribute between the cytosol and mitochondrial matrix in fundamentally different ways, i.e., according to the extent of ionization or according to the function of H+-coupled transport systems. Results show that the average cytosolic pH is considerably more alkaline than the region to which mitochondria are exposed. Because mitochondria are localized predominantly in the basal region, the results are consistent with a transcellular pH gradient within the cytosol of proximal tubule cells. Experiments analyzing the effects of inhibiting efflux of HCO3- at the basal surface and Na+-H+ exchange at the apical surface support the interpretation that the function of these systems contributes to the transcellular pH gradient. The existence of a heterogeneity in pH within the cytosol has important implications concerning the function and regulation of numerous cell processes.