AIMS: Recent studies suggest that Helicobacter pylori is an invasive enteropathogen. However, the efficiency with which this pathogen invades mammalian cells remains unknown. Therefore, this study was designed to investigate the invasion frequencies of HEp-2 cells by clinical strains of H pylori. METHODS: An acridine orange assay and cultured HEp-2 cell monolayers were used to determine the HEp-2 cell penetration frequencies of 17 clinical isolates and one American Type Culture Collection (ATCC) strain of H pylori, and single clinical strains of Yersinia enterocolitica, Shigella flexneri, and a non-invasive ATCC Escherichia coli strain. RESULTS: The acridine orange assay demonstrated that invasion frequencies of HEp-2 cells by all H pylori isolates were significant and, in most instances, exceeded those for the S flexneri strain and equalled those for the Y enterocolitica strain. The assay also showed that internalised H pylori organisms remained viable for at least six hours, the maximum time that bacteria and HEp-2 cells were co-incubated. CONCLUSIONS: These results may have important implications for treatment and prevention strategies for this gastric pathogen. Furthermore, the acridine orange assay may be useful for assessing, in vitro, the ability of conventional and newer antibiotics, alone or in combination, to kill intracellular H pylori organisms.