Adenovirus (Ad) DNA synthesis was the first replicating system for which both initiation and elongation were achieved in soluble extracts of eukaryotic cells. One of the major advantages of the Ad system is the ability to express its cloned DNA polymerase (pol) in a full functional form in vitro. This has opened the possibility of rapid and selective mutagenesis in replication protein for which at least five enzymatic reactions have been defined in vitro. Furthermore, the recent elucidation that many DNA polymerases have conserved consensus domains arranged in a linear array on the polypeptide has emphasized the common origin and shared functions between some of the pro- and eukaryotic polymerases. Site-specific mutagenesis of the Ad DNA pol and the preterminal protein (PTP), which is the primer for new Ad DNA chains, will be pursued to elucidate the active sites for the various functions of both these replicative proteins. In contrast to the extremely small amounts of Ad pol and pTP that were formerly available from large amounts of Ad infected cells, we now have considerable more of these proteins, can label them with radioactivity and can be certain that in the transfected source for each of these proteins, there is no contamination with any of the other viral-coded replication proteins. The third viral-coded protein, the ss-stranded DNA binding protein (DBP), has been cloned in an expression vector similar to Ad DNA pol and pTP and should be of considerable value in determining DBP functional domains through site specific mutation similar to the procedures used for the other 2 viral-coded proteins. With the successful cloning of all of the viral proteins necessary for Ad DNA replication it is now possible to introduce selected mutations into virtually any domain and determine the effect on the activity of each change. Since the in vitro soluble system for the replication of A DNA reproduces almost all the steps necessary for the replication of the viral genome, the activity of any mutant can be tested without the need for reincorporating the replication gene back into virions. Chimeric genes incorporating Ad and herpes simplex DNA pol will be constructed utilizing the information about the collinear, conserved, consensus sequences are by these 2 proteins. The information obtained about domains of the pols that control the selection of the DNA polymerases as targets for antiviral chemotherapy may be important the prevention and therapy of a variety of infectious diseases. The polymerases are also good targets for control of the proliferation of normal and malignant cells.