The complete sequence of pSS, which is the large virulence plasmid of Shigella sonnei, was determined. The 214-kb plasmid is composed of segments of virulence-associated genes, the O-antigen gene clusters, a range of replication and maintenance genes, and large numbers of insertion sequence (IS) elements. Two hundred and forty-one open reading frames (ORFs) were identified, of which 117 are highly homologous to IS elements or transposases, 57 are homologous to known pathogenesis-associated proteins, and 30 are related to replication, plasmid maintenance, or other metabolic functions. Thirty-seven ORFs have no similarity to proteins with a known function, including two with no significant similarity to any hypothetical proteins. Interestingly, 10 ORFs encoding O-antigen gene clusters were identified on the plasmid and this is markedly different from most other Shigella spp. virulent plasmids. A novel toxin-antitoxin system, a series of stbDE homologs, was found on the plasmid immediately downstream of the replication region; the sole segregation stability system may be responsible for the instability of pSS. The pSS plasmid is a mixture of genes with different origins and functions. The sequence suggests a remarkable history of IS-mediated recombination and acquisition of DNA across a range of bacterial species.