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A 140-megadalton plasmid (pWR110), which has previously been associated with virulence in Shigella flexneri, was transferred to Escherichia coli K-12. Segments of S. flexneri chromosomal material were then transferred to the plamid-bearing K-12 strains. The virulence of these transconjugant hybrids was assessed in the HeLa cell model, in ligated rabbit ileal loops, or in the Sereny test. A K-12 strain which harbored only pWR110 invaded HeLa cells, produced minimal lesions in the rabbit ileal mucosa, and was negative in the Sereny test. Plasmid-containing K-12 hybrids which had incorporated various shigella chromosomal regions gave differential reactions in the rabbit ileal loops and in the Sereny test. Analysis of these transconjugants indicated that three regions were linked with virulent phenotypes. These included the his region (when the genes responsible for O-antigen synthesis were cotransferred) and the kcp locus (linked to the lac-gal region). Either of these chromosomal regions was sufficient to allow invasion of the rabbit ileal mucosa. In addition to both of these regions, another shigella chromosomal segment linked to the arg and mtl loci was necessary for fluid production in the rabbit ileal loop and for a positive Sereny reaction. Thus, derivatives of an E. coli K-12 strain, constructed by the stepwise conjugal transfer of a large plasmid and three chromosomal segments from S. flexneri, appeared to contain the necessary determinants for full pathogenicity in a variety of laboratory models.