OBJECTIVE: To determine whether serum titers of anti-Epstein-Barr virus (EBV) antibodies are elevated in blood specimens collected up to 30 years prior to onset of multiple sclerosis (MS). METHODS: Individuals with MS were identified among members of the Kaiser Permanente Northern California health plan who participated in the multiphasic examinations administered between 1965 and 1974. Stored serum samples were used to compare anti-EBV antibody titers in 42 individuals who developed MS with age-matched and sex-matched controls. RESULTS: The geometric mean titers of antibodies to the Epstein-Barr nuclear antigen (EBNA) complex and its component EBNA-1 were significantly higher in the MS cases when compared with matched controls. The relative risk of MS associated with a 4-fold increase in antibody titers was 2.1 (95% confidence interval, 1.1-3.8) for the EBNA complex and 1.8 (95% confidence interval, 1.1-2.9) for EBNA-1. Elevations of antibody titers to the EBNA complex and EBNA-1 among MS cases first occurred between 15 to 20 years before the onset of symptoms and persisted thereafter. CONCLUSION: The elevation of anti-EBV titers is probably an early event in the pathogenesis of MS and is unlikely to be the result of an aspecific immune dysregulation.