The regions required for replication (Rep) and for stable maintenance (Stb) of the 230 kilobase virulence plasmid, pMYSH6000, in Shigella flexneri 2a YSH6000 were identified on the restriction enzyme Sa/l fragments, C and O, respectively. The stable plasmid, pMYSH6610, consisting of adjacent fragments C and O was found to belong to the incompatibility group Fl. In all strains of shigellae and enteroinvasive Escherichia coli (EIEC) tested, virulence plasmids were found to be highly homologous with the Rep region of pMYSH6000. The restriction map of this probe was very similar to that of the inc region of RepFIIA replicon. The virulence plasmid of S. flexneri 6, S. boydii, S. dysenteriae and EIEC all coexisted stably with pMYSH6610, whereas that of S. flexneri other than serotype 6 and S. sonnei did not. These findings indicate that all the virulence plasmids of shigellae and EIEC are highly related but can be divided into at least 2 groups.