Carbonic anhydrase (CA) IV facilitates HCO(3) reabsorption in the renal proximal tubule by catalyzing the reversible hydration of CO(2). CAIV is tethered to cell membranes via a glycosylphosphatidylinositol (GPI) lipid anchor. As there is basolateral as well as apical CAIV staining in proximal tubule, the molecular identity of basolateral CAIV was examined. Biotinylation of confluent monolayers of rat inner medullary collecting duct cells stably transfected with rabbit CAIV showed apical and basolateral CAIV, and in the cell transfectants expressing high levels of CAIV, a transmembrane form was targeted to the basolateral membrane. Basolateral expression of CAIV ( approximately 46 kDa) was confirmed in normal kidney tissue by Western blotting of vesicle fractions enriched for basolateral membranes by Percoll density fractionation. We examined the mode of membrane linkage of basolaterally expressed CAIV in the kidney cortex. CAIV detected in basolateral or apical membrane vesicles exhibited similar molecular size by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis following deglycosylation, and was equally sensitive to phosphatidylinositol-specific phospholipase C digestion, indicating that CAIV is expressed on the basolateral membrane as a GPI-anchored protein. Half of the hydratase activity of basolateral vesicles was resistant to SDS denaturation, compatible with being CAIV. Thus, GPI-anchored CAIV resides in the basolateral membrane of proximal tubule epithelia where it may facilitate HCO(3) reabsorption via association with kNBC1.