Virulence of Yersinia enterocolitica O:8 is associated with pO:8, a 42-megadalton plasmid. We constructed a mobilizable pO:8 derivative by successive in vitro and in vivo genetic manipulations. The in vitro constructed hybrid molecule pRK290B8-5 consisting of the mobilizable vector pRK290B and a 2.9-megadalton BamHI fragment of pO:8 was conjugally transferred to a Y. enterocolitica strain of serotype O:8 which harbored the virulence plasmid pO:8. From Yersinia transconjugants, a cointegrate was isolated which apparently formed by homologous recombination between the two component plasmids. The cointegrate was mobilized into plasmidless Y. enterocolitica strains of different serotypes. The transconjugants of serotype O:8 were found to express all four plasmid-associated phenotypes: (i) mouse lethality (Ml), (ii) conjunctivitis provocation in the guinea pig eye (Con), (iii) calcium requirement for growth at 37 degrees C (Mox), and (iv) agglutinogens (Ag8). The transconjugants of serotype O:3 expressed the phenotypes Con, Mox, and Ag8 but were nonlethal for mice (Ml-). The transconjugants of serotype O:5 remained avirulent for mice (Ml-) and for the guinea pig eye (Con-) but expressed the phenotypes Mox and Ag8. These data show that the virulence plasmid is probably not functionally interchangeable within different serotypes of Y. enterocolitica.