The regulator of ATPase of vacuoles and endosomes (RAVE) complex is implicated in vacuolar H(+)-translocating ATPase (V-ATPase) assembly and activity. In yeast, rav1 mutants exhibit a Vma(-) growth phenotype characteristic of loss of V-ATPase activity only at high temperature. Synthetic genetic analysis identified mutations that exhibit a full, temperature-independent Vma(-) growth defect when combined with the rav1 mutation. These include class E vps mutations, which compromise endosomal sorting. The synthetic Vma(-) growth defect could not be attributed to loss of vacuolar acidification in the double mutants, as there was no vacuolar acidification in the rav1 mutant. The yeast V-ATPase a subunit is present as two isoforms, Stv1p in Golgi and endosomes and Vph1p in vacuoles. Rav1p interacts directly with the N-terminal domain of Vph1p. STV1 overexpression suppressed the growth defects of both rav1 and rav1vph1, and allowed RAVE-independent assembly of active Stv1p-containing V-ATPases in vacuoles. Mutations causing synthetic genetic defects in combination with rav1 perturbed the normal localization of Stv1-green fluorescent protein. We propose that RAVE is necessary for assembly of Vph1-containing V-ATPase complexes but not Stv1-containing complexes. Synthetic Vma(-) phenotypes arise from defects in Vph1p-containing complexes caused by rav1, combined with defects in Stv1p-containing V-ATPases caused by the second mutation. Thus RAVE is the first isoform-specific V-ATPase assembly factor.