A 31-kb fragment of the large virulence plasmid of Shigella flexneri is necessary for bacterial entry into epithelial cells in vitro. One locus of this fragment encodes the IpaA, -B, -C, and -D proteins, which are the dominant antigens of the humoral immune response during shigellosis. To address the role of the ipa genes, which are clustered in an operon, we constructed a selectable cassette that does not affect transcription of downstream genes and used this cassette to inactivate the ipaB, ipaC, and ipaD genes. Each of these nonpolar mutants was defective in entry and lysis of the phagocytic vacuole but was not impaired in adhesion to the cells. We showed that, like IpaB and IpaC, IpaD is secreted into the culture supernatant and that none of these proteins is necessary for secretion of the other two. This result differentiates the Ipa proteins, which direct the entry process, from the Mxi and Spa proteins, which direct secretion of the Ipa proteins. Moreover, lack of either IpaB or IpaD resulted in the release of larger amounts of the other Ipa polypeptides into the culture medium, which indicates that, in addition to their role in invasion, IpaB and IpaD are each involved in the maintenance of the association of the Ipa proteins with the bacterium.