Cell-free replication of SV40 DNA in extracts prepared from S phase cells is at least 20-fold more efficient than in extracts from G1 cells. The increased activity of S phase extracts correlates with the presence of an S phase-specific cellular factor that enhances DNA unwinding at the replication origin. This change in origin-DNA structure during the initiation of SV40 replication proceeds through at least three discrete steps which can be distinguished by their extent of topologic unwinding (linking differences of −1, −2 and −5). Specific DNA elements flanking the core origin enhance replication in vivo and facilitate the formation of the pre-initiation complexes, indicating that formation of these underwound conformations may be the limiting step in the initiation of DNA synthesis. In addition, the factor that activates DNA replication in extracts from S phase cells also enhances the formation of the most highly underwound −5 pre-initiation complex. These observations suggest that during SV40 replication, formation of the rate-limiting pre-initiation complex is the focus of at least three regulatory elements. Two of these are DNA sequences flanking the replication origin and the third is a cellular factor specific to the S phase cell.