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Under anaerobic growth conditions,Escherichia colioperates a two-component signal transduction system, termed Arc, that consists of ArcB protein, a transmembrane sensor kinase and ArcA protein, the cognate response regulator. In response to low oxygen levels, autophosphorylated ArcB phosphorylates ArcA, and the resulting phosphorylated ArcA (ArcA-P) functions as a transcriptional regulator of the genes necessary to maintain anaerobic growth. Under anaerobic conditions, cells maintain a slow growth rate, suggesting that the initiation of chromosomal replication is regulated to reduce the initiation frequency. DNase I footprinting experiments revealed that ArcA-P binds to the left region of the chromosomal origin, oriC. ArcA-P did not affect thein vitroreplication of plasmid DNA containing the ColE1 origin nor the in vitroreplication of viral DNAs; however, ArcA-P specifically inhibited in vitro E. colichromosomal replication. This inhibition was caused by the prevention of open complex formation, a necessary step in the initiation of chromosomal replication. Our in vitroresults suggest that the Arc two-component system participates in regulating chromosomal initiation under anaerobic growth conditions.