Nuclei isolated from Chinese hamster ovary (CHO) cells at various times during the G1 phase of the cell cycle were stimulated to enter S phase by incubation in Xenopus egg cytosol. Replication of DNA initiated within the dihydrofolate reductase (DHFR) origin locus in nuclei isolated late in G1, but at random sites in nuclei isolated early in G1. A discrete transition point occurred 3 to 4 hours after metaphase. Neither replication licensing nor nuclear assembly was sufficient for origin recognition. Thus, a distinct cell cycle-regulated event in the nucleus restricts the initiation of replication to specific sites downstream of the DHFR gene.