The molecular mechanisms involved in DNA replication are not yet fully understood because a well defined in vitro system capable of carrying out the entire replication process of double-stranded DNA molecules has not been available. We have recently assembled the purified replication proteins coded by the bacteriophage T4 into in vitro apparatuses capable of efficient replication of double-stranded DNA templates by a process that is semiconservative, discontinuous, faithful and generates product molecules lacking rapidly renaturable hairpin areas. It is planned to use this system composed of 6 extremely pure proteins and pure DNA molecules to approach some of the unanswered problems in replication. We plan to analyze the factors involved in generating the amazingly high fidelity observed in DNA duplication. Using this system we would also determine the special features in the double-stranded DNA template that are recognized by the complex of the replication proteins resulting in the initiation of DNA synthesis. In vivo, DNA exists in a tightly folded complex with proteins and the cell membrane. Using gentle lysis conditions in the presence of low salt and counterions, I have recently isolated the intracellular T4 DNA as compact DNA-protein complexes that serve as excellent templates for in vitro DNA synthesis. This synthesis is further stimulated by the addition of the 6 purified replication proteins. Using this more natural template, we shall study the roles played by the various accessory proteins coded by the phage (the products of DNA-delay and DNA-arrest genes, some of which are membrane components) in DNA replication. It is hoped that these studies will not only lead to a clearer understanding of the mechanism of DNA-replication in prokaryotic cells but will also help us develop methods to identify and isolate proteins carrying out similar processes in eukaryotic cells.