During the course of acute Epstein-Barr virus (EBV) infection, there is a rise of oxygen radical production. As a consequence, the production of the oxygen radical scavenger manganese superoxide dismutase (MnSOD) is increased. Patients with acute EBV infections regularly develop autoantibodies against MnSOD that are able to inhibit the enzyme activity in vitro. To elucidate the origin of the autoantibodies, the epitopes on MnSOD were determined. The entire sequence of MnSOD was synthesized as overlapping pentadecapeptides, which were scanned for their reactivity with sera of patients with acute EBV infections. Sera as well as affinity-purified anti-MnSOD antibodies reacted with the peptides pno15 (amino acids 47–61) and pno30 (amino acids 122–136) lying in crucial parts of the MnSOD tetramer. The two main epitopes pno15 and pno30 showed sequence homologies with EBV-encoded proteins. Reactivity of affinity-purified antibodies with a peptide of the homologous BGLF4 points to a molecular mimicry causing the occurrence of anti-MnSOD antibodies. Anti-MnSOD antibodies were able to block the protective effects of MnSOD in a model for oxidative damage produced by xanthine/xanthine oxidase in EAhy926 endothelial cells. Thus, these autoantibodies may contribute in vivo to the clinical symptoms by accumulation of toxic oxygen radicals. J. Med. Virol. 71:408–416, 2003. © 2003 Wiley-Liss, Inc.