RPA is a cellular, three‐subunit, single‐stranded (ss) DNA binding protein, which assists T‐antigen in the assembly of the pre‐priming complex in the SV40 replication system. By immunodepletion and complementation, we have identified RPA as an essential factor for cellular DNA replication in Xenopus extracts. RPA assembles post‐mitotically on the decondensing chromosomes into numerous subnuclear pre‐replication centres (preRCs) which serve, upon formation of the nuclear membrane, as RCs for the initiation of DNA synthesis. By a variety of experiments including the use of isolated components, we demonstrate that an inactive cdc2‐cyclin B kinase complex is essential to allow post‐mitotic assembly of the preRCs. In contrast, the active cdk2‐cyclin A kinase does not impede or facilitate the assembly of preRCs. Digestion analysis using the single‐strand‐specific P1 nuclease as well as competition experiments with ssDNA, reveal that replication‐associated unwinding of the DNA, assisted by RPA, requires the formation of the nuclear membrane. The p21 cdk‐interacting protein Cip1 appears to inhibit DNA replication prior to the unwinding DNA step, but after assembly of preRC and nuclear reconstruction.