Recent experiments in budding yeast and Xenopus have provided new insights into the regulation of eukaroytic DNA replication. The multi-subunit origin recognition complex plays a key role in initiation, remaining bound at origins of replication during most of the cell cycle. Early in the cell cycle, Cdc6 and the Mcm proteins 'reset' chromatin for another round of DNA replication. Cyclin-dependent kinases appear to play a dual role, both in activating replication origins and blocking the formation of new pre-replicative complexes; thus limiting replication to once per cell cycle.