In Escherichia coli, initiation of chromosome replication requires that DnaA binds to R boxes (9-mer repeats) in oriC, the unique chromosomal replication origin. At the time of initiation, integration host factor (IHF) also binds to a specific site in oriC. IHF stimulates open complex formation by DnaA on supercoiled oriC in cell-free replication systems, but it is unclear whether this stimulation involves specific changes in the oriC nucleoprotein complex. Using dimethylsulphate (DMS) footprinting on supercoiled oriC plasmids, we observed that IHF redistributed prebound DnaA, stimulating binding to sites R2, R3 and R5(M), as well as to three previously unidentified non-R sites with consensus sequence (A/T)G(G/C) (A/T)N(G/C)G(A/T)(A/T)(T/C)A. Redistribution was dependent on IHF binding to its cognate site and also required a functional R4 box. By reducing the DnaA level required to separate DNA strands and trigger initiation of DNA replication at each origin, IHF eliminates competition between strong and weak sites for free DnaA and enhances the precision of initiation synchrony during the cell cycle.