Adenovirus DNA polymerase is one of three viral proteins and two cellular proteins required for replication of the adenovirus genome. During initiation of viral DNA synthesis the viral DNA polymerase transfers dCMP onto the adenovirus preterminal protein, to which it is tightly bound. The domain structure of the 140 kDa DNA polymerase has been probed by partial proteolysis and the sites of proteolytic cleavage determined by N-terminal sequencing. At least four domains can be recognised within the DNA polymerase. Adenovirus preterminal protein interacts with three of the four proteolytically derived domains. This was confirmed by cloning and expression of each of the individual domains. These data indicate that, like other members of the pol alpha family of DNA polymerases, the adenovirus DNA polymerase has a multidomain structure and that interaction with preterminal protein takes place with non-contiguous regions of the polypeptide chain over a large surface area of the viral DNA polymerase.