The double par locus of Escherichia coli virulence factor pB171 consists of two adjacent and oppositely oriented par loci of different types, called par1 and par2. par1 encodes an actin ATPase (ParM), and par2 encodes an oscillating, MinD-like ATPase (ParA). The par loci share a central cis-acting region of approximate to 200 bp, called parC1, located between the two par loci. An additional cis-acting region, parC2, is located downstream of the parAB operon of par2. Here we show that ParR of par1 and ParB of par2 bind cooperatively to unrelated sets of direct repeats in parC1 to form the cognate partition and promoter repression complexes. Surprisingly, ParB repressed transcription of the noncognate par operon, indicating cross-talk and possibly epistasis between the two systems. The par promoters, P1 and P2, affected each other negatively. The DNA binding activities of ParR and ParB correlated well with the observed transcriptional regulation of the par operons in vivo and in vitro. Integration host factor (IHF) was identified as a novel factor involved in par2-mediated plasmid partitioning.