The relationship between gp140, the membrane C3d receptor (CR2) of human B lymphocytes, and the Epstein-Barr virus receptor (EBVR) was analyzed by using the polyclonal anti-gp140, previously prepared by immunizing rabbits with highly purified gp140 (isolated by some of us) from CR2/EBVR-positive Raji cells. Polyclonal anti-gp72, a C3-binding membrane component, not related to the EBVR but also expressed on the Raji cell surface, was used as a control. Binding of rabbit IgG and EBV on cells was assessed by using immunofluorescence techniques with analysis by flow cytofluorometry. A semiquantitative bioassay was also used to measure the EBV binding. Polyclonal monospecific anti-gp140 IgG inhibits directly the binding of EBV to Raji cells at the same concentration that inhibits the binding of EC3d on cells, whereas a 35 times higher concentration of anti-gp72 IgG or preimmune serum IgG does not. Anti-gp140 IgG treatment also inhibits the induction of EBV-determined nuclear antigen in normal tonsil B lymphocytes or in EBV-negative Ramos cells, whereas high concentrations of anti-gp72 IgG or preimmune serum IgG have no effect. These data strongly suggest that gp140, the CR2 of human B lymphocytes, is also the EBVR.