We have recently cloned and characterized a unique sodium bicarbonate cotransporter, NBC3, which unlike other members of the NBC family, is ethylisopropylamiloride (EIPA) inhibitable, DIDS insensitive, and electroneutral (A. Pushkin, N. Abuladze, I. Lee, D. Newman, J. Hwang, and I. Kurtz. J. Biol. Chem.274: 16569–16575, 1999). In the present study, a specific polyclonal antipeptide COOH-terminal antibody, NBC3-C1, was generated and used to determine the pattern of NBC3 protein expression in rabbit kidney. A major band of ∼200 kDa was detected on immunoblots of rabbit kidney. Immunocytochemistry of rabbit kidney frozen sections revealed specific staining of the apical membrane of intercalated cells in both the cortical and outer medullary collecting ducts. The pattern of NBC3 protein expression in the collecting duct was nearly identical to the same sections stained with an antibody against the vacuolar H⁺-ATPase 31-kDa subunit. In addition, the NBC3-C1 antibody coimmunoprecipitated the vacuolar H⁺-ATPase 31-kDa subunit. Functional studies in outer medullary collecting ducts (inner stripe) showed that type A intercalated cells have an apical Na⁺-dependent base transporter that is EIPA inhibitable and DIDS insensitive. The data suggest that NBC3 participates in H⁺/base transport in the collecting duct. The close association of NBC3 and the vacuolar H⁺-ATPase in type A intercalated cells suggests a potential structural/functional interaction between the two transporters.