A considerable effort is being made to discover more effective anticancer agents and to develop rapid, accurate, and reliable methods for evaluating the usefulness of the new compounds and analyzing their mechanisms of action. In this regard, our laboratory was the first to report the isolation, characterization, and extensive purification of an intact, stable, and fully functional mammalian cell multiprotein complex, which we designated the DNA synthesome. The DNA synthesome has been isolated from a wide variety of mammalian cells as well as from tissues and has been shown to be fully competent to support SV40 origin-specific and large T antigen-dependent DNA replication in vitro. In this article we will review the utilization of the DNA synthesome as a powerful in vitro model system for analyzing the mechanism of action of S-phase specific anticancer agents.