Abstract: We describe herein the isolation and purification of a multi-protein complex for DNA replication from MDA MB-468 human breast cancer cells as well as human breast tumor tissue and xenografts from nude mice injected with human breast cancer cell line MCF-7. This complex, designated the "DNA synthesome", fully supports the in vitro replication of simian virus 40 (SV40) origin-containing DNA in the presence of the viral large T-antigen. Since the SV40 virus utilizes the host's cellular proteins for its own DNA replication, our results indicate that the DNA synthesome plays a role not only in viral DNA synthesis but in human breast cell DNA replication as well. Our studies demonstrate that the following DNA proteins are incorporated into the DNA synthesome: DNA polymerase .alpha., DNA primase, DNA polymerase .delta., proliferating cell nuclear antigen (PCNA), replication protein A (RP-A), replication protein C (RF-C), DNA topoisomerase I and II, and DNA polymerase .epsilon..