The Epstein-Barr virus (EBV) DNA polymerase (Pol) holoenzyme is an essential enzyme required for ori-Lyt dependent EBV DNA replication. Using singly primed M13ssDNA circles as template, the EBV DNA Pol holoenzyme synthesized DNA chains greater than the unit length of M13 ssDNA in addition to full length products even at a low ratio of polymerase molecule per templates. The long replication products consisted of circular double-stranded DNA with single-stranded tails that were sensitive to mung bean nuclease. Reconstitution of the EBV Pol holoenzyme by preincubation of BALF5 Pol catalytic subunit and BMRF1 Pol accessory subunit in vitro resulted in reproduction of the strand displacement DNA synthesis. Thus, the EBV DNA Pol holoenzyme by itself is able to produce strand displacement coupled to the polymerization process in a highly processive way in the absence of any other protein.