An environmental bacterial isolate, Iso10, previously found to show serological cross-reactivity with type-specific Shigella boydii 15 antisera was subjected to further molecular and serological analyses that revealed interspecies transfer of the O antigen gene cluster. Western blot analysis of Iso10 cell surface extracts and purified lipopolysaccharides demonstrated strong cross-reactivity with S. boydii 15-specific monovalent antisera and a lipopolysaccharide gel banding profile similar to that of S. boydii 15. Biochemical and phylogenetic analyses identified the Iso10 isolate as Escherichia fergusonii. O antigen gene cluster analyses of Iso10, carried out by restriction fragment length analysis of the amplified ~10-kb O antigen-encoding gene cluster, revealed a profile highly similar to that of S. boydii 15, confirming the presence of the S. boydii 15 somatic antigen in Iso10. To the best of our knowledge, this is the first report of interspecies transfer of O antigen-encoding genes between S. boydii and E. fergusonii, and it has implications for our understanding of the role of lateral gene transfer in the emergence of novel Shigella serotypes.