About 50% of Helicobacter pylori isolates produce a vacuolating toxin in vitro, which may be an important determinant of virulence. Because ammonium salts potentiate H. pylori toxin activity, the effect of other weak bases upon toxin activity was determined. Vacuolation of HeLa cells was quantitated using a neutral red uptake assay. As expected, ammonium chloride, trimethylamine, triethanolamine, and nicotine each induced vacuolation of HeLa cells when tested independently. In addition, each of these weak bases potentiated H. pylori vacuolating toxin activity, whereas sodium chloride or sodium hydroxide did not. Sequential incubation of cells with toxin followed by nicotine resulted in potentiation of vacuolation, whereas sequential incubation in the reverse order did not lead to potentiation. Monensin inhibited the formation of vacuoles by either H. pylori vacuolating toxin or nicotine. The potentiation of H. pylori toxin activity by ammonia and nicotine may contribute to gastroduodenal mucosal injury associated with this infection.