We have previously cloned a 44 kb fragment from the virulence plasmid of Shigella flexneri serotype 5 strain M90T which is capable of restoring invasiveness to an avirulent, plasmidless mutant. This report presents a genetic and physical analysis of Tn5 mutations in recombinant clone pHS4108. Tn5 mutagenesis allowed identification of at least five regions implicated in the entry phenotype. These regions were located on a 20 kb portion of pHS4108. Expression of the insertion mutants was studied by immunoblots using the serum of a convalescent monkey infected by S. flexneri 2a, which recognized four plasmid-associated polypeptides. We propose that the four immunogenic polypeptides, a, b, c, and d, are encoded an operon.