We describe a cell-free system from HeLa cells that initiates DNA replication under cell cycle control. G1 but not G2 phase nuclei initiate replication when coincubated with S phase nuclei in cytosolic extracts from S phase but not from G1 or G2 phase HeLa cells. S phase nuclei or an S phase nuclear extract are required for the initiation of semiconservative DNA replication in G1 nuclei but not for elongation in S phase nuclei. S phase nuclear extract could be replaced by recombinant human cyclins A and E complexed to Cdk2 but not by Cdk2 alone or by human cyclin B1 complexed to Cdc2. In S phase cytosol, cyclins A/Cdk2 and E/Cdk2 triggered initiation synergistically.