Simian virus 40 (SV40) DNA replication dependent on the SV40 origin of replication and the SV40 large tumor (T) antigen has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, these components included the DNA polymerase alpha-primase complex, topoisomerase I, and a fraction that contained a single-stranded DNA binding protein. The latter protein, which sediments at 5.1 S on glycerol gradients and copurifies with two major protein species of 72 and 76 kDa, was isolated solely by its ability to support SV40 DNA replication. The purified system retained the species-specific DNA polymerase alpha-primase requirement previously observed with crude fractions; the complex from HeLa cells supported SV40 replication, whereas that from calf thymus and mouse cells did not. DNA containing the polyomavirus origin of replication was replicated in a system containing polyomavirus T antigen, the HeLa single-stranded DNA binding protein-containing fraction, and DNA polymerase alpha-primase complex from mouse, but not HeLa, cells. While crude fractions yielded closed circular duplex DNA, none was detected with the purified system. Nevertheless, the addition of a crude fraction to the purified system yielded closed circular monomer products.