The effects of topoisomerases I and II on the replication of SV40 DNA were examined using an in vitro replication system of purified proteins that constitutes the monopolymerase system. In the presence of the two topoisomerases, two distinct nascent DNAs were formed. One product arising from the replication of the leading template strand was approximately half the size of the template DNA, whereas the other product derived from the lagging template strand consisted of short DNAs. These products were synthesized from both SV40 naked DNA and SV40 chromosomes. For the replication of SV40 naked DNA, either topoisomerase I or II maintained replication fork movement and supported complete leading strand synthesis. When SV40 chromosomes were replicated with the same proteins, reactions containing only topoisomerase I produced shorter leading strands. However, mature size DNA products accumulated in reactions supplemented with topoisomerase II, as well as in reactions containing only topoisomerase II. In the presence of crude extracts of HeLa cells, VP-16, a specific inhibitor of topoisomerase II, blocked elongation of the nascent DNA during the replication of SV40 chromosomes. These results indicate that topoisomerase II plays a crucial role as a swivelase in the late stage of SV40 chromosome replication in vitro.