Abstract: Yeast mutants lacking V-ATPase subunits (vma mutants) were sensitive to several different oxidants in a recent genomic screen (Thorpe et al (2004) PNAS 101:6564-9). We confirmed that mutants lacking a V1 subunit (vma2), Vo subunit, or either of the two Vo a subunit isoforms are acutely sensitive to H2O2, and more sensitive to menadione and diamide than wild-type cells. The vma2 mutant contains elevated levels of reactive oxygen species and high levels of oxidative protein damage even in the absence of an applied oxidant, suggesting an endogenous source of oxidative stress. vma2 mutants lacking mitochondrial DNA showed neither improved growth nor decreased sensitivity to peroxide, excluding respiration as the major source of the endogenous reactive oxygen species in the mutant. Double mutants lacking both VMA2 and components of the major cytosolic defense systems exhibited synthetic sensitivity to H2O2. Microarray analysis comparing wild-type and vma2 mutant cells grown at pH 5, permissive conditions for the vma2 mutant, indicated high level upregulation of several iron uptake and metabolism genes that are part of the Aft1/Aft2 regulon. TSA2, which encodes an isoform of the cytosolic thioredoxin peroxidase, was strongly induced, but other oxidative stress defense systems were not induced. The results indicate that V-ATPase activity helps to protect cells from endogenous oxidative stress.