Murine gamma-herpesvirus 68 (MHV-68) is a model for the study of the pathogenesis of gamma-herpesviruses. Epstein-Barr virus (EBV) is a highly related gamma-herpesvirus that causes significant disease in humans. The major membrane antigen gp350 of EBV is a candidate vaccine antigen for protection against EBV-related disease. An MHV-68 glycoprotein, gp150, has significant homology to EBV gp350. We have therefore used the MHV-68 gp150 to model the potential efficacy of EBV gp350 in protecting from virus-associated disease. A recombinant vaccinia virus expressing MHV-68 gp150 was constructed. This recombinant vaccinia virus was used to infect mice via the subcutaneous route. This vaccination resulted in production of MHV-68-neutralising antibodies. Mice were then challenged intra-nasally with MHV-68. MHV-68-associated mononucleosis was virtually abrogated in immunised mice. However, mice did establish MHV-68 latency. The results suggest that gp350 may be effective as an immunogen to prevent EBV-associated infectious mononucleosis in humans that are EBV-seronegative.