The unwinding of the origin of replication (oriC) is a critical step in initiation of DNA replication inEscherichia coli.Previous observations indicate that efficient unwinding of supercoiled plasmid templates containingoriCsequences requires the DnaA initiation protein and one or more accessory factors. The precise contribution of each protein to this process is unknown. Here, we demonstrate that unwinding can occur under physiological conditions at the same bases inoriC,in either the presence or the absence of initiation proteins, as detected by a single-strand specific nuclease, P1. This suggests thatoriCunwinding is a spontaneous event determined solely by DNA sequence. DnaA and IHF, as part of a large nucleoprotein complex, may function to stabilize the DNA strand opening prior to initiation of DNA replication.