Mounting evidence indicates that infection with Epstein–Barr virus (EBV) plays a major role in the pathogenesis of multiple sclerosis (MS). Defective elimination of EBV-infected B cells by CD8⁺ T cells might cause MS by allowing EBV-infected autoreactive B cells to accumulate in the brain. Here we undertake a comprehensive analysis of the T cell response to EBV in MS, using flow cytometry and intracellular IFN-γ staining to measure T cell responses to EBV-infected autologous lymphoblastoid cell lines and pools of HLA-class-I-restricted peptides from EBV lytic or latent proteins and cytomegalovirus, in 95 patients and 56 EBV-seropositive healthy subjects. In 20 HLA-A2⁺ healthy subjects and 20 HLA-A2⁺ patients we also analyzed CD8⁺ T cells specific for individual peptides, measured by binding to HLA-peptide complexes and production of IFN-γ, TNF-α and IL-2. We found a decreased CD8⁺ T cell response to EBV lytic, but not cytomegalovirus lytic, antigens at the onset of MS and at all subsequent disease stages. CD8⁺ T cells directed against EBV latent antigens were increased but had reduced cytokine polyfunctionality indicating T cell exhaustion. During attacks the EBV-specific CD4⁺ and CD8⁺ T cell populations expanded, with increased functionality of latent-specific CD8⁺ T cells. With increasing disease duration, EBV-specific CD4⁺ and CD8⁺ T cells progressively declined, consistent with T cell exhaustion. The anti-EBNA1 IgG titre correlated inversely with the EBV-specific CD8⁺ T cell frequency. We postulate that defective CD8⁺ T cell control of EBV reactivation leads to an expanded population of latently infected cells, including autoreactive B cells.