Infection of mice with a neurotropic strain of MHV (MHV-A59), a non-neurotropic strain of MHV (MHV-2), and a set of recombinant viruses (kindly provided by Dr. Michael Lai) were used to map genetic determinants of viral neurotropism and demyelination. Following intracerebral (IC) inoculation of 4-week old C57B1/6 mice, 1LD50 of MHV-A59 produced acute meningoencephalitis and hepatitis, and subsequently chronic CNS demyelinating disease. IC inoculation of 1LD50 of MHV-2 produced acute hepatitis without CNS disease. Recombinants ML-3, ML-11, ML-7, ML-8, ML-9 and ML-10 produced acute encephalitis similar to MHV-A59. According to previous oligonucleotide fingerprinting analysis the only common denominator of the neurotropic recombinant viruses was an M gene derived from MHV-A59. Sequencing of PCR-amplified viral S and M genes confirmed that the M genes of neurotropic viruses are derived from A59 while the S genes of neurotropic viruses are either derived from MHV-2 or from A59. In tissue culture, ML-11, ML-3 and MHV-2 are fusion negative, while A59, ML-7, ML-8 and ML-10 are fusion positive. Thus, neurotropism in MHVs is not linked to fusion or the S gene. Moreover, the M gene may be a significant determinant of neurotropism and acute encephalitis.