Entry of Epstein-Barr virus (EBV) into B lymphocytes requires the binding of viral glycoprotein 42 (gp42), a C-type lectin family member, to HLA class II. Recently, the structure of the gp42:HLA-DR1 complex was determined. In order to confirm the interaction as determined in the structural study and to identify other potential interactive residues, a mutational analysis of HLA class II was performed. A secreted form of gp42 (sgp42) reacted with a conformation-specific monoclonal antibody and blocked EBV infection. The binding of sgp42 and EBV entry to two sets of HLA class II mutants were tested. The first set of mutants were based on the known interaction of the C-type lectin Ly49A with HLA class I, and the second set of mutants were based on the identified interface in the gp42:HLA-DR1 complex. As expected, none of the mutants that would be predicted to interfere with the interaction of Ly49A with class I affected the interaction of gp42 with HLA class II, whereas mutants in amino acids identified in the gp42:HLA-DR1 structure inhibited sg42 binding to class II. In general, sgp42 binding correlated with efficient entry of EBV, as demonstrated by the necessity of glutamic acid 46 or arginine 72 in class II molecules. Furthermore, other HLA class II residues buried within the interface of gp42 and HLA class II when mutated had either no effect or a decrease in both binding and entry and implicate a region of class II important in stabilizing the interaction with gp42. These studies provide insight into the entry and fusion processes of the critical interaction between gp42 and HLA class II.