Extracts of Xenopus eggs efficiently initiate and complete chromosomal DNA replication in vitro, under normal cell cycle controls. Such extracts can be depleted of Xenopus p34cdc2, either by affinity depletion using the protein p13suc1 or by specific immunodepletion. Depleted extracts are incapable of initiating DNA replication, although they efficiently elongate replication forks initiated in undepleted extracts. Depletion of p34cdc2 does not prevent nuclear assembly, which is required for the initiation of DNA replication in this system. Activity can be restored to depleted extracts by readdition of p13suc1 eluates enriched for p34cdc2. These results demonstrate that p34cdc2, or a very closely related protein, is involved in the initiation of chromosomal DNA replication in the cell cycle of higher eukaryotes.