The Epstein-Barr virus (EBV) glycoprotein gp85 is the EBV gH homologue and is thought to be involved in penetration of virus through the B-cell membrane. However, although the glycoprotein is functionally important, it is found in very low abundance in infected cells and in the virion. To facilitate analysis of the structure and function of gp85, recombinant vaccinia viruses were constructed to express the glycoprotein. Recombinant gp85 was recognized by polyclonal antibody made to a peptide derived from the gp85 sequence, but not by monoclonal antibodies that reacted with the native molecule. Unlike native gp85, the recombinant protein contained no sugars that were resistant to endoglycosidase H and it was not transported to the cell surface. The native protein was found to be associated with two additional glycoproteins with apparent M(r) of 25,000 and 42,000. Antibody made to a peptide derived from a sequence in the BKRF2 open reading frame immunoprecipitated glycoproteins with the mobilities of gp85 and its associated 25,000-Da molecule. These data suggest that the BKRF2 gene product, like that encoded by its positional homologues gL of herpes simplex virus and the UL115 gene product of human cytomegalovirus, associates with gp85 and may be required for glycoprotein processing.