Viral infections are though to play an important role in the pathogenesis of multiple sclerosis (MS) potentially through molecular mimicry. An identical sequence was found in both myelin basic protein (MBP, residues 96–102), a candidate autoantigen for MS, and human herpesvirus‐6 (HHV‐6 U24, residues 4–10) that is a suspected viral agent associated with MS. In this study, we showed that greater than 50% of T cells recognizing MBP_93‐105 cross‐reacted with and could be activated by a synthetic peptide corresponding to residues 1 to 13 of HHV‐6 U24 in MS patients. The estimated precursor frequency of these cross‐reactive T cells recognizing both peptides, MBP_93‐105 and HHV‐6 (U24)_1‐13, was significantly elevated in MS patients compared with that in healthy controls. These cross‐reactive CD4+ T cells represented the same Th1 phenotype as that of monospecific T cells recognizing MBP_93‐105. There were increased antibody titers for both peptide HHV‐6 (U24)_1‐13 and peptide MBP_93‐105 in the same patients with MS compared with those in healthy controls, suggesting B‐cell sensitization to the antigens in MS patients. The study provides important evidence in the understanding of the potential role of HHV‐6 infection/reactivation in the activation of autoimmune reactivity to MBP and its implication in the pathogenesis of MS. Ann Neurol 2003