OBJECTIVES: To study in relapsing-remitting (RR) multiple sclerosis (MS) whether exacerbations and brain activity as measured by magnetic resonance imaging (MRI) are associated with plasma levels of anti-Epstein Barr (EBV) antibodies and EBV DNA. METHODS: This was a prospective study with 73 RR MS patients followed for an average of 1.7 years with frequent neurological examination and blood sampling. Antibodies to various EBV proteins were measured by ELISA and plasma EBV DNA was measured by PCR. RESULTS: All MS patients had IgG antibodies to EBV (viral capsid antigen (VCA) and/or EBV nuclear antigen (EBNA)), irrespective whether samples were taken at stable disease or exacerbation. A significantly elevated percentage of the patients (48%) had antibodies against EBV antigens (early antigen, EA) that indicate active viral replication, compared with the age matched healthy controls (25%). Antibodies against a control herpesvirus, cytomegalovirus, were similar between the two groups. The percentage of EA positive individuals and EA titres did not differ between stable disease or exacerbation. Anti-VCA IgM was positive in three cases, unrelated to disease activity. Using a highly sensitive PCR on 51 samples taken at exacerbation visits, only three patients were found to have one timepoint with viraemia, and this viraemia was unrelated to disease activity. Of special note was the fact that anti-EA seropositive patients remained seropositive during follow up, with stable titres over time. We hypothesised that these patients may constitute a subgroup with higher disease activity, due to the triggering effect of a chronic attempt of the virus to reactivate. The EA positive group did not differ from the EA negative with respect to clinical disease activity or other characteristics. However, in the EA positive group, analysis with gadolinium enhanced MRI indicated more MRI disease activity. CONCLUSIONS: There was no evidence for increased clinical disease activity in the subgroup of MS patients with serological signs of EBV reactivation. However, the observation that chronic EBV reactivation may be associated with increased inflammatory activity as assessed by gadolinium enhanced MRI lesions should be reproduced in a larger and independent dataset.