Mammalian chromosomal domains replicate at defined, developmentally regulated times during S phase. The positions of these domains in Chinese hamster nuclei were established within 1 hr after nuclear envelope formation and maintained thereafter. When G1 phase nuclei were incubated in Xenopus egg extracts, domains were replicated in the proper temporal order with nuclei isolated after spatial repositioning, but not with nuclei isolated prior to repositioning. Mcm2 was bound both to early- and late-replicating chromatin domains prior to this transition whereas specification of the dihydrofolate reductase replication origin took place several hours thereafter. These results identify an early G1 phase point at which replication timing is determined and demonstrate a provocative temporal coincidence between the establishment of nuclear position and replication timing.