MHV-A59 produces acute encephalitis, acute hepatitis and chronic demyelination in infected mice. MHV-2 produces only hepatitis and mild meningitis but without encephalitis or demyelination. We have previously studied a set of recombinant viruses between these two strains. The common denominator of viruses that produced encephalitis was a membrane (M) gene derived from MHV-A59. Thus to study the potential contribution of the M gene to acute encephalitis, chimeric viruses were produced in which the M gene of MHV-A59 was substituted with the M gene of MHV-2 by targeted recombination. A control virus was produced in which the M gene of A59 was recombined back into an A59 background. Viruses were then analyzed for their biologic properties and compared with the phenotypes of MHV-A59 and MHV-2 by histopathology and plaque assays for viral titers in organs following intracerebral (IC) inoculation. All three chimeric viruses had a phenotype similar to MHV-A59. Thus, the replacement of the M gene of MHV-A59 with that of MHV-2 is insufficient to produce a phenotype that lacks encephalitis similar to MHV-2.