In this report we describe, for the first time, the purification and characterization of a replication-competent multiprotein form of DNA polymerase (designated the DNA synthesome) from the human leukemia cell line (HL-60) using a series of centrifugation, ion-exchange chromatography and velocity sedimentation steps. The proteins and enzymatic activities thus far identified to co-purify with the leukemia cell DNA synthesome include the DNA polymerases alpha and delta, DNA primase, proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), replication protein A (RP-A), and DNA topoisomerases I and II. We have demonstrated that the DNA synthesome is fully competent to replicate simian virus 40 (SV40) replication origin containing DNA in vitro in the presence of the viral large T-antigen. This result implies that all of the cellular activities required for large T-antigen-dependent in vitro SV40 DNA synthesis are present in the isolated human leukemia cell DNA synthesome. Since SV40 is extensively dependent on the host cell's DNA synthetic machinery for its own DNA replication, our results indicate that the isolated leukemia cell DNA synthesome may play a role not only in viral DNA synthesis but also in human leukemia cell DNA replication. We recently proposed a model to represent the DNA synthesome that was isolated from HeLa and murine cells. Our data indicate that the organization of the DNA synthesome from HL-60 cells also fits this proposed model. The purified DNA synthesome will not only allow the further study of the molecular mechanisms required to carry out human leukemia cell DNA replication, but may also provide a tool for eventually dissecting some of the regulatory controls of the cell's DNA synthetic machinery.