The VacA toxin secreted by Helicobacter pylori is considered to be an important virulence factor in the pathogenesis of peptic ulcer disease and gastric cancer. VacA monomers self-assemble into water-soluble oligomeric structures and can form anion-selective membrane channels. The goal of this study was to characterize VacA-VacA interactions that may mediate assembly of VacA monomers into higher order structures. We investigated potential interactions between two domains of VacA (termed p-33 and p-55) by using a yeast two-hybrid system. p-33/p-55 interactions were detected in this system, whereas p-33/p-33 and p-55/p-55 interactions were not detected. Several p-33 proteins containing internal deletion mutations were unable to interact with wild-type p-55 in the yeast two-hybrid system. Introduction of these same deletion mutations into the H. pylori vacA gene resulted in secretion of mutant VacA proteins that failed to assemble into large oligomeric structures and that lacked vacuolating toxic activity for HeLa cells. Additional mapping studies in the yeast two-hybrid system indicated that only the N-terminal portion of the p-55 domain is required for p-33/p-55 interactions. To characterize further p-33/p-55 interactions, we engineered an H. pylori strain that produced a VacA toxin containing an enterokinase cleavage site located between the p-33 and p-55 domains. Enterokinase treatment resulted in complete proteolysis of VacA into p-33 and p-55 domains, which remained physically associated within oligomeric structures and retained vacuolating cytotoxin activity. These results provide evidence that interactions between p-33 and p-55 domains play an important role in VacA assembly into oligomeric structures.