C12, an attenuated, fusion delayed, very weakly hepatotropic mutant of mouse hepatitis virus strain A59 (MHV-A59) has been further characterized. We have previously shown that C12 has two amino acid substitutions relative to wild type virus in the spike protein, Q159L (within a region of S1 shown to bind to viral receptor in an in vitro assay) and H716D (in the proteolytic cleavage recognition site). We have sequenced the rest of the 31-kb genome of C12 and compared it to wild type virus. Only three additional amino acids substitutions were found, all encoded within the replicase gene. Analysis of C12 in vivo in C57BI/6 mice has shown that despite the fact that this virus replicates in the brain to titers at least as high as wild type and causes acute encephalitis similar to wild type, this virus causes a minimal level of demyelination and only at very high levels of virus inoculation. Thus acute encephalitis is not sufficient for the induction of demyelination by MHV- A59. Analysis of mutants isolated at earlier times from the same persistently infected glial cell culture as C12, as well as mutants isolated from a second independent culture of persistently infected glial cells, suggests that both the weakly demyelinating and the weakly hepatotropic phenotypes of C12 are associated with the Q159L amino acid substitution.