Studies of Epstein-Barr virus (EBV) infection and pathogenesis are limited by the lack of recombinant EBV bearing a reporter gene. Currently such studies typically utilize cellular transformation and immortalization as indicators of virus infection which takes several weeks. To investigate the dependence on known cellular receptors for EBV infection, a novel enhanced green fluorescent protein (EGFP) expressing virus, designated EBfaV-GFP, was used to infect a variety of different cell types. EBfaV-GFP infects B lymphoma-derived cell lines, lymphoblastoid cell lines (LCLs) and primary B cells, as judged by expression of EGFP. Ability of clonal cells to be infected with EBfaV-GFP correlates with expression of the known EBV entry mediators CD21 and HLA-DR. EGFP-expressing LCLs were derived by infection of primary B cells with EBfaV-GFP. Cells of the Jurkat line, derived from T lymphocytes, could not be infected, and infected primary lymphocytes did not include appreciable numbers of CD2-positive cells, showing that EBV rarely infects T cells. Expression of EGFP by EBV provides the opportunity to rapidly visualize infection in living cells and better delineate populations of infected cells.