Abstract: Screening the Saccharomyces cerevisiae homozygous diploid deletion library against a sublethal concentration of cisplatin revealed seventy-six strains sensitive to the drug. As expected, the largest category of deletes, representing 40% of the sensitive strains, was composed of strains lacking genes involved in DNA replication and damage repair. Deletes lacking function of the highly conserved vacuolar H(+) translocating ATPase (V-ATPase) comprised the category representing the second largest number of sensitive strains. The effect on cell death exhibited by V-ATPase mutants was found to be a general response to various DNA damaging agents as opposed to being specific to cisplatin, as evidenced by sensitivity of the mutants to a DNA alkylating agent, hydroxyurea and UV irradiation. Loss of V-ATPase does not affect DNA repair, as double mutants defective for V-ATPase function and DNA repair pathways were more sensitive to cisplatin than the single mutants. V-ATPase mutants are more prone to DNA damage than wild type cells, indicated by enhanced activation of the DNA damage checkpoint. Vacuole function per se is not cisplatin sensitive, as vacuolar morphology and vacuolar acidification were unaffected by cisplatin in wild type cells. V-ATPase also controls cytoplasmic pH, so the enhanced sensitivity to DNA damage may be associated with the drop in pHi associated with V-ATPase mutants. The increased loss in cell viability induced by cisplatin at lower pH in V-ATPase mutants supports this hypothesis. The loss in viability seen in wild type cells under the same conditions was far less dramatic.