ABSTRACTP1parfamily members promote the active segregation of a variety of plasmids and plasmid prophages in gram-negative bacteria. Each has genes for ParA and ParB proteins, followed by aparSpartition site. The large virulence plasmid pWR100 ofShigella flexnericontains a new P1parfamily member: pWR100par. Although typicalparAandparBgenes are present, the putative pWR100parSsite is atypical in sequence and organization. However, pWR100parSpromoted accurate plasmid partition inEscherichia coliwhen the pWR100 Par proteins were supplied. Unique BoxB hexamer motifs withinparSdefine species specificities among previously described family members. Although substantially different from P1parSfrom the P1 plasmid prophage ofE. coli, pWR100parShas the same BoxB sequence. As predicted, the species specificity of the two types proved identical. They also shared partition-mediated incompatibility, consistent with the proposed mechanistic link between incompatibility and species specificity. Among several informative sequence differences between pWR100parSand P1parSis the presence of a 21-bp insert at the center of the pWR100parSsite. Deletion of this insert left much of theparSactivity intact. Tolerance of central inserts with integral numbers of helical DNA turns reflects the critical topology of these sites, which are bent by binding the host IHF protein.