Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS) with features suggestive of T-cell-mediated pathology. Most prior reports have focused on CD4⁺ T cells with the underlying assumption that MS is predominantly a CD4⁺ T helper 1 (Th1)-mediated disease. In this report, we used a novel flow cytometric approach to evaluate autoreactive T-cell responses against a large variety of neuroantigenic targets. We found that both CD4⁺ and CD8⁺ T cells targeted against several CNS autoantigens were widely prevalent in patients with MS and healthy individuals. Whereas the distribution of CD4⁺ responses was similar in different groups, patients with relapsing-remitting MS showed a higher proportion of CNS-specific CD8⁺ responses. Autoreactive CD4⁺ T cells from patients with MS exhibited a more differentiated Th1 phenotype compared with healthy subjects. Similarly, CNS-specific CD8⁺ T-cell responses from patients with MS were functionally distinct from those in healthy individuals. Collectively, these studies reveal the high prevalence of class I-restricted autoreactive CD8⁺ T-cell responses in MS that has been underappreciated thus far. The results emphasize the need to evaluate both CD4⁺ and CD8⁺ T-cell responses in MS and to make both subsets a consideration in the development of novel therapeutic strategies. (Blood. 2004; 103:4222-4231)