Several of the 13 subunits comprising mammalian H⁺-ATPases have multiple alternative forms, encoded by separate genes and with differing tissue expression patterns. These may play an important role in the intracellular localization and activity of H⁺-ATPases. Here we report the cloning of a previously uncharacterized human gene, ATP6V0E2, encoding a novel H⁺-ATPase e-subunit designated e2. We demonstrate that in contrast to the ubiquitously expressed gene encoding the e1 subunit (previously called e), this novel gene is expressed in a more restricted tissue distribution, particularly kidney and brain. We show by complementation studies in a yeast strain deficient for the ortholog of this subunit, that either form of the e-subunit is essential for proper proton pump function. The identification of this novel form of the e-subunit lends further support to the hypothesis that subunit differences may play a key role in the structure, site and function of H⁺-ATPases within the cell.