An Epstein-Barr virus (EBV) specific monoclonal antibody (MAb), designated 55H3, was produced after immunizing BALB/c mice with 12-O-tetradecanoylphorbol-13-acetate (TPA) activated B95-8 cells. It was possible to demonstrate that this MAb detected an EBV-specific antigen(s) in the EBV genome-positive producer cell lines B95-8 and M-ABA by immunofluorescence. Immunoglobulin prepared from ascites fluid neutralized the activity of the EBV-encoded DNA polymerase, but not the alkaline DNase. By Western blotting, the 55H3 MAb reacts with an 85/80-KD polypeptide. The 55H3 should be useful in examining the role of EBV DNA polymerase in viral replication.