A major 135-kDa DNA binding protein (mDBP) encoded by the BALF2 open reading frame of Epstein-Barr Virus (EBV) is known to be an essential protein for the induction of the lytic cycle. The present investigation was carried out to know whether this protein forms a complex in vivo with other viral DNA binding proteins (DBP) involved in DNA replication: DNA polymerase, EA-D (diffused early antigen), and DNAase. Immunoprecipitation assays followed by mono- and two-dimensional electrophoresis showed that mDBP forms a complex with these three DBP. Other complexes were also found such as EA-D/DNAase, DNA polymerase/DNAase, and DNA polymerase/EA-D. The complexed forms already exist in the early stage of EBV cycle before DNA synthesis is induced in the EBV producer P3HR-1 cell line. The exonuclease activity encoded by DNAase was found to be inhibited when this enzyme complexed with mDBP, while the EBV DNA polymerase retained its activity in the complexed form with mDBP. Our results suggest that these complexes already present before DNA synthesis are necessary for EBV DNA synthesis.