In G1-arrested cells infected between 1 and 12 h after having been stimulated by fresh serum to progress to S phase, polyoma virus DNA synthesis proceeded in the first half of S phase, and virus and whole cellular DNA accumulated at about the same time. However, in cells infected later than 14 h after serum stimulation, virus DNA synthesis was shifted to the next S phase. Thus, a permissive cell attains competence for polyoma virus DNA replication at a precise moment during an S phase initiated by fresh serum, which can efficiently replace the early virus host DNA stimulation function. When cells were incubated in serum that had lost its capacity to stimulate host DNA synthesis by pre-absorption with growing cells, normal yields of polyoma DNA could nevertheless be observed, which shows that extensive replication of host DNA does not seem to be an obligatory condition for virus DNA replication.