We have inserted a 311-base pair DNA fragment containing the simian virus 40 (SV40) origin of DNA replication, the early promoter, and the tumor (T) antigen binding sites into a bacterial plasmid and cloned it. This recombinant plasmid, pSV01, binds to a purified T antigen in vitro and replicates in monkey cells when supplied with large T antigen. A series of deletion mutations was generated in the origin sequences of pSV01 DNA by mutagenesis in vitro. The replication of these mutant DNAs in monkey cells was compared with their ability to bind to purified D2 protein. Mutant DNAs deficient in binding to D2 protein also exhibit reduced levels of replication in monkey cells. These findings provide biochemical evidence that the initiation of SV40 DNA synthesis may involve a direct interaction of T antigen with sequences at the origin of replication.