Complement receptor type 2 (CR2/CD21) is essential for the attachment of Epstein-Barr virus (EBV) to the surface of B-lymphocytes in an interaction mediated by the viral envelope glycoprotein gp350. The heavily glycosylated structure of EBV gp350 has recently been elucidated by X-ray crystallography and the CR2 binding site on this protein characterized. To identify the corresponding gp350 binding site on CR2, we have undertaken a site-directed mutagenesis study targeting regions of CR2 that have previously been implicated in the binding of CR2 to the C3d/C3dg fragments of complement component C3. Wild-type or mutant forms of CR2 were expressed on K562 cells, and the ability of these CR2-expressing cells to bind gp350 was measured using flow cytometry. Mutations directed towards the two N-terminal extracellular domains of CR2 (SCR1-2) reveal that a large contiguous surface of CR2 SCR1-2 is involved in gp350 binding, including a number of positively charged residues (R13, R28, R36, K41, K57, K67 and R83). These data appear to complement the CR2 binding site on gp350 which is characterized by a preponderance of negative charge. In addition to identifying the importance of charge in the formation of a CR2 – gp350 complex we also provide evidence that both SCR1 and SCR2 make contact with gp350. Specifically, two anti-CR2 monoclonal antibodies, designated as mAb 171 and 1048 and whose primary epitopes are located within SCR2, inhibit binding of wild-type CR2 to EBV gp350; with regard to SCR1, both K562 cells expressing an S15P mutation and recombinant S15P CR2 proteins exhibit diminished gp350 binding.