Approximately 50% of Helicobacter pylori strains produce a toxin in vitro that induces vacuolation of eukaryotic cells. To determine whether ion transport pathways are important in the formation of toxin-induced vacuoles, HeLa cells were incubated with H. pylori toxin in the presence of nine different inhibitors of ion-transporting ATPases. Oligomycin, an inhibitor of predominantly F1F0-type ATPases, had no effect on toxin activity. Inhibitors of predominantly V-type ATPases, exemplified by bafilomycin A1, inhibited the formation of vacuoles in response to the H. pylori toxin and reversed the vacuolation induced by the toxin. In contrast, at concentrations of > or = 100 nM, ouabain and digoxin, inhibitors of the Na(+)-K+ ATPase, potentiated the activity of H. pylori toxin. The inhibitory effects of bafilomycin A1 could not be overcome by the potentiating effects of ouabain. These data suggest that intact activity of the vacuolar ATPase of eukaryotic cells is a critical requirement in the pathogenesis of cell vacuolation induced by H. pylori toxin and that vacuole formation by this toxin is associated with altered cation transport within eukaryotic cells.