Previously, we showed that the transmembrane (M) and surface (S) glycoproteins were recognized by splenic CD4 T lymphocytes harvested from mice infected intraperitoneally with mouse hepatitis virus, strain JHM (MHV-JHM), whereas only the S protein was recognized by splenocytes derived from mice with MHV-induced chronic demyelination. From these results, it could not be determined which proteins were recognized by T cells localized in the infected central nervous system (CNS). Herein, we show that CD4 T cells responding to both the M and S proteins can be detected in the CNS of mice with either acute encephalitis or the chronic demyelinating disease. As part of these analyses, two CD4 T cell epitope regions encompassing residues 328-347 and 358-377 within the S protein were identified. Both epitopes, as well as a previously identified M-specific epitope, were recognized by the CNS-derived lymphocytes. Finally, viral RNA harvested from mice with chronic demyelination was analyzed for mutations in the S specific CD4 T cell epitopes since changes resulting in escape from CD8 T cell surveillance were previously identified in these samples. A mutation in epitope region S(328-347) (ala to thr at position 337) was detected in a minority of samples but this change did not abrogate recognition of the epitope and therefore was unlikely to contribute to virus persistence. In conclusion, these studies identify epitopes recognized by MHV-specific CD4 T cells in the infected CNS and show that these cells are preferentially located at the site of infection in mice with clinical disease.