Temperature-sensitive mutants in the N complementation group of human adenovirus type 5 are defective at the nonpermissive temperature for replication of virus DNA and for transformation of rat embryo cells. We show that nuclear extracts prepared from Ad5ts 149-infected cells grown at the nonpermissive temperature fail to replicate DNA in vitro. The defect lies in the first step in the initiation of viral DNA synthesis, the formation of a covalent linkage between the terminal protein precursor (pTP) and dCMP. A 140 kilodalton (140 kd) protein which complements these defective extracts and contains DNA polymerase activity has been purified from HeLa cells infected with wild-type Ad2. It is tightly associated with the 80 kd pTP in a replication complex. Both of these proteins are products of the E2B region of the adenovirus genome, and the 140 kd protein coding sequences lie immediately downstream from those encoding the 80 kd protein. These results demonstrate that adenovirus encodes a novel DNA polymerase that is required for priming of DNA synthesis at the origin of replication. This protein may also function in the initiation of transformation of cultured cells.