Initiation of DNA replication at oriC occurs bidirectionally both in vivo and in vitro. Although the proteins involved in establishing the replication forks are known, little is known about the events that ensure that initiation is bidirectional. We show here that in the absence of DNA gyrase, replication fork progression from oriC on a plasmid template in vitro is unidirectional, although both replication forks have formed at the origin. There was no bias in the release of one fork or the other, ruling out protein blockage of one fork as a possible reason for the asymmetric release. Timely release of both forks required the presence of either DNA gyrase or topoisomerase IV, suggesting that modulation of the topology of the origin region is the governing factor.