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The gp350 glycoprotein encoded by BLLF1 is crucial for efficient Epstein-Barr virus (EBV) infection of resting B cells. Gp350 binds to CD21, but whether this interaction sums up its functions remains unknown. We generated gp350-null EBVs that display CD19-, CD21-, or CD22-specific antibodies at their surface (designated as {Delta}BLLF1-Ab). Gp350-complemented ({Delta}BLLF1-C) and {Delta}BLLF1-Ab were found to bind equally well to B cells. Surprisingly, {Delta}BLLF1 binding was reduced only 1.7-fold relative to its complemented counterparts. Furthermore, B cells exposed to {Delta}BLLF1-Ab or {Delta}BLLF1 viruses presented structural antigens with comparable efficiency and achieved 25 to 80% of the T-cell activation elicited by {Delta}BLLF1-C. These findings show that the gp350-CD21 interaction pair plays only a modest role during virus transfer to the endosomal compartment. However, primary B cells or Raji B cells infected with {Delta}BLLF1-C viruses displayed a 35- to 70-fold higher infection rates than those exposed to {Delta}BLLF1, {Delta}BLLF1-CD22Ab, or {Delta}BLLF1-CD19Ab viruses. Complementation of the gp350 knockout phenotype with CD21Ab substantially enhanced infection rates relative to {Delta}BLLF1 but remained sevenfold (Raji B-cell line) to sixfold (primary B cells) less efficient than with gp350. We therefore infer that gp350 mainly exerts its functions after the internalization step, presumably during release of the viral capsid from the endosomal compartment, and that CD21-dependent but also CD21-independent molecular mechanisms are involved in this process. The latter appear to be characteristic of B-cell infection since transfection of CD21 in 293 cells improved the infection rates with both {Delta}BLLF1-CD21Ab and {Delta}BLLF1-C to a similar extent.