In eukaryotes the entire genome is replicated precisely once in each cell cycle. No DNA is re-replicated until passage through mitosis into the next S-phase. We have used a cell-free DNA replication system from Xenopus eggs to determine which mitotic changes permit DNA to re-replicate. The system efficiently replicates sperm chromatin, but no DNA is re-replicated in a single incubation. This letter shows that nuclei replicated in vitro are unable to re-replicate in fresh replication extract until they have passed through mitosis. However, the only mitotic change which is required to permit re-replication is nuclear envelope permeabilization. This suggests a simple model for the control of DNA replication in the cell cycle, whereby an essential replication factor is unable to cross the nuclear envelope but can only gain access to DNA when the nuclear envelope breaks down at mitosis.