Sodium-independent Cl movement (i.e., Cl-anion exchange) has not previously been identified in the basolateral membranes of colonic epithelial cells. The present study demonstrates Cl-HCO₃ exchange as the mechanism for ³⁶Cl uptake in basolateral membrane vesicles (BLMV) prepared in the presence of a protease inhibitor cocktail from rat distal colon. Studies of ³⁶Cl uptake performed with BLMV prepared with different types of protease inhibitors indicate that preventing the cleavage of C-terminal end of AE2 protein by serine-type proteases was responsible for the demonstration of Cl-HCO₃ exchange. In the absence of voltage clamping both outward OH-gradient (pHout/pHin: 7.5/5.5) and outward HCO₃-gradient stimulated transient ³⁶Cl uptake accumulation. However, voltage clamping with K-ionophore, valinomycin, almost completely (88%) inhibited the OH gradient-driven ³⁶Cl uptake, while HCO₃ gradient-driven ³⁶Cl uptake was only partially inhibited (40%). Both electroneutral HCO₃ and OH gradient-driven ³⁶Cl uptake were: 1) completely inhibited by DIDS, an anion exchange inhibitor, with a half maximal inhibitory constant (Ki) of approximately 26.9 and 30.6 µM, respectively; 2) not inhibited by NPPB, a Cl channel blocker; 3) saturated by increasing extravesicular Cl concentration with a Km for Cl of approximately 12.6 and 14.2 mM, respectively; and 4) present in both surface and crypt cells. Intracellular pH (pH_i) was also determined with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxy-fluorescein-acetomethylester (BCECF-AM) in an isolated superfused crypt preparation. Removal of Cl resulted in a DIDS-inhibitable increase in pH_i both in HCO₃-buffered and in the nominally HCO₃-free buffered solutions (0.28 ± 0.02 and 0.11 ±0.02 pH units, respectively). We conclude that a carrier-mediated electroneutral Cl-HCO₃ exchange is present in basolateral membranes and that, in the absence of HCO₃, Cl-HCO₃ exchange can function as a Cl-OH exchange and regulate pHi across basolateral membranes of rat distal colon.