The Escherichia coli chromosomal origin contains several bindings sites for factor for inversion stimulation (FIS), a protein originally identified to be required for DNA inversion by the Hin and Gin recombinases. The primary FIS binding site is close to two central DnaA boxes that are bound by DnaA protein to initiate chromosomal replication. Because of the close proximity of this FIS site to the two DnaA boxes, we performed in situ footprinting with 1, 10-phenanthroline-copper of complexes formed with FIS and DnaA protein that were separated by native gel electrophoresis. These studies show that the binding of FIS to the primary FIS site did not block the binding of DnaA protein to DnaA boxes R2 and R3. Also, FIS appeared to be bound more stably to oriC than DnaA protein, as deduced by its reduced rate of dissociation from a restriction fragment containing oriC . Under conditions in which FIS was stably bound to the primary FIS site, it did not inhibit oriC plasmid replication in reconstituted replication systems. Inhibition, observed only at high levels of FIS, was due to absorption by FIS binding of the negative superhelicity of the oriC plasmid that is essential for the initiation process.