The soluble replication system is which the exogenously added simian virus 40 (SV40) DNA can be replicated semiconservatively in vitro, has been developed (Ariga and Sugano, J.Virol. 48, 481, 1983). This paper further characterized the in vitro products synthesized on the cloned DNA containing the origin of SV40 DNA replication. The time course and pluse-chase experiments showed that the in vitro products were converted from the open circle to closed circles having the various superhelical densities, and finally to the twisted formI DNA seen in vivo by the analysis of agarose gel electrophoresis, alkaline sucrose gradient centrifugation, and density-transfer in isopycnic centrifugation. The replicative intermediates isolated after the short term incubation had replicated strands of the size smaller than the full length, most of which correspond to that of the putative Okazaki fragment. These and the previous results indicate that this in vitro system should be useful to investigate the molecular mechanism of SV40 DNA replication.