Vacuolar H(+)-ATPases (V-ATPases) are highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. Although it is generally believed that V-ATPases transport protons by a rotary catalytic mechanism analogous to that used by F(1)F(0)-ATPases, the structure and subunit composition of the central or peripheral stalk of the multisubunit complex are not well understood. We searched for proteins that bind to the E subunit of V-ATPase using the yeast two-hybrid assay and identified the H subunit as an interacting partner. Physical association between the E and H subunits of V-ATPase was confirmed in vitro by precipitation assays. Deletion mapping analysis revealed that a 78-amino acid fragment at the amino terminus of the E subunit was sufficient for binding to the H subunit. Expression of the amino-terminal fragments of the E subunits from human and yeast as dominant-negative mutants resulted in dramatic decreases in bafilomycin A(1)-sensitive ATP hydrolysis and proton transport activities of V-ATPase. Our data demonstrate the physiological significance of the interaction between the E and H subunits of V-ATPase and extend previous studies on the arrangement of subunits on the peripheral stalk of V-ATPase.