The Escherichia coli chromosomal origin consists of a duplex-unwinding region and a region bearing a DNA-bending protein, IHF-binding site, and clusters of binding sites for the initiator protein DnaA. ATP-DnaA molecules form highly organized oligomers in a process stimulated by DiaA, a DnaA-binding protein. The resultant ATP-DnaA complexes promote local unwinding of oriC with the aid of IHF, for which specific interaction of DnaA with the single-stranded DNA is crucial. DnaA complexes also interact with DnaB helicases bound to DnaC loaders, promoting loading of DnaB onto the unwound DNA strands for bidirectional replication. Initiation of replication is strictly regulated during the cell cycle by multiple regulatory systems for oriC and DnaA. The activity of oriC is regulated by its methylation state, whereas that of DnaA depends on the form of the bound nucleotide. ATP-DnaA can be yielded from initiation-inactive ADP-DnaA in a timely manner depending on specific chromosomal DNA elements termed DARS (DnaA-reactivating sequences). After initiation, DnaA-bound ATP is hydrolyzed by two systems, yielding ADP-DnaA. In this review, these and other mechanisms of initiation and its regulation in E. coli are described.