The nucleotide sequence of a 4.8 kilobase (kb) HindIII fragment from pWR100, the virulence plasmid of Shigella flexneri 5, was determined and analysed. This fragment encodes polypeptides b (62 kilodalton, kD) and c (43 kD) which have already been described as two of the four immunogenic polypeptides of Shigellae. The nucleotide sequence revealed that in addition to the ipaB and ipaC genes encoding polypeptides b and c, a third complete open reading frame was found within the fragment. The gene, named ippI, encoded a 17 kD polypeptide. The deduced amino acids sequence of polypeptides b and c showed no signal peptide but presence of highly hydrophobic domains compatible with a transmembraneous location. The surprising A and T richness of the three genes as compared with the Escherichia coli and Shigella genomes, resulted in a biased codon usage, and raises the question of the origin of the sequences.