It is shown here that plasmids containing the replication origin of Escherichia coli (oriC) cannot replicate in an extrachromosomal state in E. coli cells with the polA1hip3 double mutation. This E. coli mutant is deficient in the polymerizing function of DNA polymerase I (Pol I) and is unable to produce functional IHF protein. The inability of the oriC minichromosomes to replicate in the absence of IHF is dependent on the absence of Pol I; cells with the polA+himA- or polA+hip- mutation, which are deficient in the alpha and beta subunits of the IHF heterodimer, respectively, can support replication of the oriC replicons. We propose that IHF-deficient cells utilize an alternative pathway of the DNA replication in which Pol I is required. In vitro DNA binding assays revealed that the IHF binding site resides between the oriC coordinates 110 and 122 and is adjacent to the DnaA "box" 1. Within the area protected by IHF we found at least 1 out of 11 GATC methylation sites present in oriC. The consequences of lack of IHF protein binding to the oriC and the indirect effects of the IHF deficiency on the oriC replication are discussed.