In vitro DNA replication system derived from eukaryotes is capable of replicating exogenous plasmid DNA containing SV40 origin of replication efficiently and accurately. Such an in vitro DNA replication system has been established. In the presence of exogenous SV40 large T antigen, shuttle-vector plasmid pZ189 could replicate efficiently in monkey kidney vero cytoplasmic extracts, which provided all the other necessary proteins for DNA replication. This in vitro DNA replication system provides a useful measure for studying the composition and function of eukaryotic DNA replication system.