We have developed a cell-free system from frog eggs that efficiently initiates and completes a single round of semi-conservative replication. 70-100% of sperm chromatin and up to 40% of plasmid DNA molecules are completely replicated in vitro. Before DNA is replicated it is assembled into nuclei surrounded by a double unit membrane studded with nuclear pores. Flow cytometry shows that initiation events are co-ordinated within individual nuclei, although different nuclei can start to replicate at different times in the same extract. This demonstrates the importance of nuclear structure in the control of DNA replication in this system. Only a single round of semi-conservative replication occurs in the cell-free system. This mirrors the way that only one round of DNA replication occurs in each cell cycle in vivo. When replicated nuclei are transferred to fresh extract they are unable to undergo another round of replication. However, if the nuclear envelope is permeabilised before nuclei are transferred to fresh extract, the DNA becomes capable of undergoing a further round of semi-conservative replication. These results suggest a simple model for the control of DNA replication within the cell cycle, whereby an essential initiation factor can only gain access to DNA when the nuclear envelope breaks down during mitosis.