Shigella flexneri kills macrophages through apoptosis, involving the induction of host cell DNA fragmentation and characteristic morphological changes. Shigella can only cause damage if it escapes from the phagolysosome into the cytoplasm. The S. flexneri cytotoxic genes have been localized to the ipa operon of shigella's virulence plasmid. ipaB, C and D deletion mutants are not invasive and therefore not cytotoxic. In order to distinguish genes involved in the escape from the phagolysosome as distinct from cytotoxicity, we constructed Shigella strains that secrete low amounts of Escherichia coli haemolysin (hlylow). These strains can escape into the cytoplasm of the macrophage even in the absence of the invasion plasmid as verified by electron microscopy and resistance to chloroquine. Macrophages were infected with different ipa mutants expressing hlylow. Both δipaC hlylow and δipaD hlylow were cytotoxic whilst δipaB hlylow and a hlylow strain cured of shigella's pathogenicity plasmid were not. Furthermore, both δipC ahlylow and δipaD hlylow killed through apoptosis as shown by both changes in ultrastructural morphology and fragmentation of the host ceil DNA. These results demonstrate that ipaB is essential for S. flexneri to induce apoptosis in macrophages.