Hybridomas producing antibodies against Epstein-Barr virus (EBV) have been produced. They have been screened for production of antibodies against the EBV-associated membrane antigens that are present on EBV producer cell lines. For the screening test we used a double-antibody radioimmunoassay with 125I-labeled rabbit anti-mouse Ig as the second step. Positive cultures were cloned and one such clone, C1, was used for further study. C1 antibody neutralizes EBV in vitro and immunoprecipitates both gp350 (the major glycoprotein in B95-8 cells) and gp220 (the major glycoprotein in P3HR-1 cells) from the membrane antigen complex, suggesting that these molecules are antigenically related. The presence of gp350/220 both on the plasma membrane and in the cytoplasm may be detected by immunofluorescence with C1 antibody.