An antibody-secreting hybrid cell line was produced by fusion of mouse myeloma cells with splenocytes from mice immunized with virions of the B95-8 strain of Epstein-Barr Virus (EBV). The monoclonal IgG antibody was shown to have anti-EBV activity by the following criteria: (i) It reacted with the membranes and the cytoplasm of seven different EBV-producing lines, but with no nonproducing line. (ii) The individual cells identified by the murine antibody were shown to be the same cells identified by a human serum having anti-EBV activity. (iii) The antibody significantly reduced the infectivity of two independent strains of EBV (namely, P3HR1K and B95-8). The antigen being recognized was characterized by immunoprecipitations of radiolabeled EBV-producer cell lysates. A single glycoprotein with an estimated molecular weight of 250,000 was identified. It is concluded that neutralization of EBV can be achieved by an IgG-class monoclonal antibody directed against a single antigenic site on a 250,000-dalton glycoprotein, which is a constituent of the EBV virion.