Involvement of adenovirus DNA polymerase (AdPol) in viral DNA replication was initially demonstrated using two temperature-sensitive (ts) adenoviruses, Ad5ts36 and Ad5ts149. Ad5ts36 is particularly interesting because a single mutation mapping to Leu391-->Phe in the AdPol gene confers ts phenotype not only for DNA replication in human cells but also for transformation in rat cells. In order to elucidate the mechanism(s) involved in the ts phenotype, we introduced the ts36 mutation in AdPol of adenovirus type 2 and overexpressed the protein using recombinant vaccinia virus expression system in order to study its biochemical properties. The results show that the ts36 AdPol is defective in the in vitro DNA replication initiation and DNA polymerase elongation assays if the protein is expressed at the nonpermissive temperature (37 degrees) independent of whether the assays are performed at 32 or 37 degrees. In contrast, the ts36 AdPol expressed at 32 degrees had activities similar to that of the wild-type protein when assayed at either temperature. Furthermore, unlike the wild-type protein, ts36 AdPol expressed at 37 degrees failed to recognize the viral DNA replication origin, but bound to a single-stranded DNA cellulose column with greater affinity, suggesting that the defect in the ts36 AdPol for DNA replication can be attributed to its altered DNA-binding properties.