Epstein-Barr virus (EBV) infection has been thought to be a key environmental factor in the development of multiple sclerosis (MS). The aim of this study is to investigate the association of EBV infection with neuromyelitis optica (NMO). We measured levels of serum antibodies against EBV antigens, including anti-viral capsid antigen (VCA) IgM, anti-VCA IgG, anti-early antigen (EA) IgM, anti-EA IgG and anti-EBV nuclear antigen-1 IgG, in 50 patients with NMO (including 12 partial form with antiaquaporin 4 antibodies), 51 patients with MS, and 52 healthy controls, and cerebrospinal fluid (CSF) antibodies in 37 patients with NMO and 33 patients with MS with ELISA. Compared with patients with MS and normal participants, patients with NMO more frequently had serum anti-EA IgG antibodies (52%), indicating more active viral replication than patients with MS (26%) and controls (25%). The antibody titres were significantly higher in the NMO group than in the MS (p=0.005) and control (p=0.005) groups. The CSF antibody titres were also higher in patients with NMO than in those with MS (p=0.03). Our results raise the hypothesis that persistent, active EBV replication is present in NMO, and may contribute to the immunological alterations that play a pathogenetic role in the disorder. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.