Coronaviruses, mouse hepatitis virus (MHV) strains, exhibit various degrees of neurotropism and hepatotropism following intracerebral (IC) infection of 4-week-old C57Bl/6 mice. Whereas MHV-A59 produces acute meningitis, encephalitis, hepatitis, and chronic demyelination, a closely related strain, MHV-2, produces only acute meningitis and hepatitis. We previously reported that the spike glycoprotein gene of MHV contains determinants of demyelination and hepatitis. To further investigate the site of demyelination and hepatitis determinants within the S gene, we sequenced the S gene of several nondemyelinating recombinant viruses. We found that three encephalitis-positive, demyelination-negative, hepatitis-negative recombinant viruses have an MHV-A59-derived S gene, which contains three identical point mutations (I375M, L652I, and T1087N). One or more of the sites of these mutations in the MHV-A59 genome are likely to contribute to demyelination and hepatitis.