Infection of mice with the JHM strain of mouse hepatitis virus (MHV) results in an acute encephalomyelitis associated with primary demyelination of the central nervous system. Efforts at understanding the components of the immune response in the development of chronic MHV-induced demyelination have implicated the antibody response and both the CD4+ and CD8+ T cell responses. In this report, we demonstrate that Balb/c (H-2d) mice immunized with the JHM (JHMV) strain of MHV develop a CD8+ cytotoxic T lymphocyte (CTL) response. One population of these virus-specific CTL recognize the nucleocapsid (N) protein. Recombinant vaccinia viruses expressing either the entire N protein or carboxy-terminal deletions were used to determine the number and location of the epitope(s) recognized. The CTLs were found to recognize a peptide contained within the carboxy-terminal 149 amino acids of the N protein. Analysis of infected cell lines expressing transfected major histocompatibility genes demonstrated that the anti-N protein CTLs were restricted exclusively to the Ld molecule. These data provide the first definition of a MHV-specific CTL response directed to a viral protein and suggest that the anti-N protein CTL response is one potential mechanism used by the host to clear JHMV from the central nervous system.