Escherichia coli is a model system to study the mechanism of DNA replication and its regulation during the cell cycle. One regulatory pathway ensures that initiation of DNA replication from the chromosomal origin, oriC, is synchronous and occurs at the proper time in the bacterial cell cycle. A major player in this pathway is SeqA protein and involves its ability to bind preferentially to oriC when it is hemi-methylated. The second pathway modulates DnaA activity by stimulating the hydrolysis of ATP bound to DnaA protein. The regulatory inactivation of DnaA function involves an interaction with Hda protein and the beta dimer, which functions as a sliding clamp for the replicase, DNA polymerase III holoenzyme. The datA locus represents a third mechanism, which appears to influence the availability of DnaA protein in supporting rifampicin-resistant initiations.