A functional role of class II antigen in the binding of Epstein-Barr virus (EBV) was deduced from the study of membrane proteins on Jijoye, an EBV receptor (EBVR)-positive B cell line, and its mutant, EBVR-negative daughter cell line, P3HR-1. From gel electrophoresis of radiolabeled microsomal membrane proteins and immunoprecipitates, we identified class II antigen on Jijoye but not on P3HR-1 cells and the presence of Ii on both cell lines. The role of these molecules in EBVR function was tested by antibody blocking of virus adsorption. Anti-p23,30 serum (to class II antigen) was found to block binding of EBV to B lymphoblasts under conditions in which normal rabbit serum, rabbit antiserum to butyrate-treated P3HR-1 cells (with ample anti-Ii antibodies), and rabbit anti-p44,12 (to class I antigen and beta 2-microglobulin) serum did not block virus binding. Only one of four commercial monoclonal antibodies (MoAb) to framework epitopes on class II antigens blocked binding of EBV, whereas all four MoAb demonstrated immunofluorescent reactivity with the EBVR+ Raji cells. In previous studies of binding of EBV to hairy leukemic cells, a substantial subpopulation of HLA-DR+, EBVR- cells was identified, in addition to HLA-DR+, EBVR+ cells. These findings were consistent with the view that the HLA-DR complex has a role in the binding of EBV but that other components are also needed for the expression of EBVR function.