Simian virds 40 (SV40) has a relatively simple and well defined genome, and is particularly appropriate as a model for studying mammalian chromatin. A nuclear system was developed for studying in vitro replication of SV40 chromatin, and the effects of cytoplasmic fractions obtained from uninfected CV-1 cells and calf thymus histones on the DNA replication were examined. The major polymerase involved in the replicative DNA synthesis was DNA polymerase α. Product analysis of replicated DNA showed that the amounts of the replicating intermediates and the open circular form of SV40 DNA (Form II) increased by in vitro DNA synthesis conducted without the addition of cytoplasmic fractions. By the addition of cytoplasmic fractions, the twisted form of SV40 DNA (Form I) as well as its replicative form and Form II were synthesized. Replicative DNA synthesis was inhibited by the addition of histone Hl but not by the addition of core histones, when the nuclear DNA to added hiatone ratio was similar to the DNA-histone ratio in eukaryotic chromatin.