A monoclonal antibody (MAb), designated 3E8, was produced against the Epstein-Barr virus BHRF1 which is a viral homologue of the anti-apoptotic protein Bcl-2. The MAb recognized the BHRF1 protein in extracts from EBV-containing cell lines after activation and EBV-negative cell lines transfected by the BHRF1 gene. Epitope mapping by Western blot analysis revealed that the antibody bound region encompassing amino acid residues 28-33 of the BHRF1. In addition to immunoblotting, the MAb could be applied widely in detection of the BHRF1 in many assays, including immunofluorescence assay, immunohistochemistry, enzyme-linked immunosorbent assay and immunoprecipitation. Most of all, when used in immunoprecipitation experiments, the MAb 3E8 showed a better effect than the existing anti-BHRF1 MAbs since radioactive isotopes were not required to intensify signals of its target antigen. Based on its great use in a variety of immunological reactions, it is a powerful tool to elucidate the biological functions of BHRF1.