DnaA protein of Escherichia coli acts in the initiation of chromosomal replication to bind to sequences in the chromosomal origin. On binding, it promotes the assembly of other replication proteins that serve to prime DNA replication and assemble the replication apparatus for bidirectional replication fork movement. A collection of monoclonal antibodies to DnaA protein have been produced, one of which is described here, that interferes with the action of DnaA protein in promoting formation of a prepriming complex. On the analysis of this process, the antibody appears to interfere with the physical interaction between DnaA and DnaB protein in the DnaB.DnaC complex. Cross-linking studies confirm that DnaA and DnaB proteins interact directly. These results provide the first direct evidence that one of the roles of DnaA protein is to act as a site for binding of DnaB protein to the DNA and perhaps orients DnaB helicase to account for the directionality of replication fork movement.