The 56-kDa B1 subunit of the vacuolar H(+)ATPase has a C-terminal DTAL amino acid motif typical of PDZ-binding proteins that associate with the PDZ protein, NHE-RF (Na(+)/H(+) exchanger regulatory factor). This B1 isoform is amplified in renal intercalated cells, which play a role in distal urinary acid-base transport. In contrast, proximal tubules express the B2 isoform that lacks the C-terminal PDZ-binding motif. Both the B1 56-kDa subunit and the 31-kDa (E) subunit of the H(+)ATPase are pulled down by glutathione S-transferase NHE-RF bound to GSH-Sepharose beads. These subunits associate in vivo as part of the cytoplasmic V1 portion of the H(+)ATPase, and the E subunit was co-immunoprecipitated from rat kidney cytosol with NHE-RF antibodies. The interaction of H(+)ATPase subunits with NHE-RF was inhibited by a peptide derived from the C terminus of the B1 but not the B2 isoform. NHE-RF colocalized with H(+)ATPase in either the apical or the basolateral region of B-type intercalated cells, whereas NHE-RF staining was undetectable in A-intercalated cells. In proximal tubules, NHE-RF was located in the apical brush border. In contrast, H(+)ATPase was concentrated in a distinct membrane domain at the base of the brush border, from which NHE-RF was absent, consistent with the expression of the truncated B2 subunit isoform in this tubule segment. The colocalization of NHE-RF and H(+)ATPase in B- but not A-intercalated cells suggests a role in generating, maintaining, or modulating the variable H(+)ATPase polarity that characterizes the B-cell phenotype.