SUMMARYSubunit a of V-ATPase in the yeast Saccharomyces cerevisiae, in contrast to its other subunits, is encoded by two genes VPH1 and STV1. While disruption of any other gene encoding the V-ATPase subunits results in growth arrest at pH 7.5, null mutants of Vph1p or Stv1p can grow at this pH. We used a polyclonal antibody to yeast Stv1p and a commercially available monoclonal antibody to Vph1p for analysis of yeast membranes by sucrose gradient fractionation, and two different vital dyes to characterize the phenotype of vph1 ▵ and stv1 ▵mutants as compared to the double mutant and the wild-type cells. Immunological assays of sucrose gradient fractions revealed that the amount of Stv1p was elevated in the vph1 ▵ strain, and that vacuoles purified by this method with no detectable endosomal contamination contain an assembled V-ATPase complex, but with much lower activity than the wild type. These results suggest that Stv1p compensates for the loss of Vph1p in the vph1 ▵ strain. LysoSensor Green DND-189 was used as a pH sensor to demonstrate unexpected changes in vacuolar acidification in stv1▵ as the Vph1p-containing V-ATPase complex is commonly considered to acidify the vacuoles. In the vph1 ▵ strain, the dye revealed slight but definite acidification of the vacuole as well. The lipophilic dye FM4-64 was used as an endocytic marker. We show that the null V-ATPase mutants, as well as the vph1 ▵ one, markedly slow down endocytosis of the dye.