To determine the role of carbonic anhydrase (CA) in colonic electrolyte transport, we studied Car-20mice, mutants deficient in cytosolic CA II. Ion fluxes were measured under short-circuit conditions in an Ussing chamber. CA was analyzed by assay and Western blots. In Car-20mouse colonic mucosa, total CA activity was reduced 80% and cytosolic CA I and membrane-bound CA IV activities were not increased. Western blots confirmed the absence of CA II in Car-20mice. Normal mouse distal colon exhibited net Na+and Cl−absorption, a serosa-positive PD, and was specifically sensitive to pH. Decrease in pH stimulated active Na+and Cl−absorption whether it was caused by increasing solution [Formula: see text], reducing [Formula: see text] concentration, or reducing pH in CO2/[Formula: see text]-free HEPES-Ringer solution. Membrane-permeant methazolamide, but not impermeant benzolamide, at 0.1 mM prevented the effects of pH. Car-20mice exhibited similar basal transport rates and responses to pH and CA inhibitors. We conclude that basal and pH-stimulated colonic electrolyte absorption in mice requires CA I. CA II and IV may have accessory roles.