SummaryThe invasive phenotype ofShigella flexneriis conferred by a 220 kb virulence plasmid, pWR100, that encodes both the Ipa proteins, which are involved in the entry process, and factors which are required for the export and correct localization of the Ipa proteins. We have characterized the mxiD gene, whose expression, like that of theipaoperon, is regulated by temperature. After inactivation ofmxiD, the mutant strain was unable to invade HeLa cells and to provoke keratoconjunctivitis in guinea‐pigs. Analysis of culture supernatants indicated that wild‐typeS. flexnerisecretes about nine polypeptides and that secretion of several of these, including IpaA, IpaB, and IpaC, is abolished in themxiDmutant. Examination of the membrane proteins of the wild‐type andmxiDstrains suggested that MxiD is an outer membrane protein. Amino acid sequence comparison revealed that MxiD is homologous to the YscC protein ofYersinia enterocoliticaand to theC‐terminal region of the PulD protein ofKlebsiella pneumoniae.Both YscC and PulD are involved in extracellular protein secretion. These results indicate that MxiD is an essential component of the Ipa secretion ap