Approximately 50% of Helicobacter pylori isolates produce a cytotoxin in vitro that induces vacuolation of eukaryotic cells. To determine the in vivo relevance of this phenomenon, we sought to detect cytotoxin-neutralizing antibodies in sera from H. pylori-infected persons. As a group, sera from 29 H. pylori-infected patients neutralized the activity of the purified cytotoxin to a significantly greater extent than sera from 24 uninfected persons (P = 0.007). The cytotoxin neutralizing activity in sera from H. pylori-infected persons was mediated predominantly by the purified IgG fraction. Sera from H. pylori-infected persons neutralized the cytotoxins produced by multiple H. pylori strains, but failed to neutralize trimethylamine-induced cell vacuolation. Neutralization of cytotoxin activity by human or immune rabbit sera was associated with immunoblot IgG recognition of an 87-kD H. pylori protein. Similarly, neutralization of the toxin by sera was associated with IgG recognition of the purified cytotoxin in an enzyme-linked immunosorbent assay (P less than 0.0001). The presence of cytotoxin-neutralizing antibodies in sera from H. pylori-infected persons indicates that the cytotoxin is synthesized in vivo.