The BamHI-Z-encoded Epstein-Barr virus (EBV) replication activator (ZEBRA) is a key mediator of the switch from latency to productive cycle in EBV virus. Antibodies against ZEBRA are a marker of EBV reactivation and are regularly found among patients with infectious mononucleosis (IM) or nasopharyngeal carcinoma (NPC), but are only rarely found among healthy EBV-seropositive donors. In order to define the serologically reactive epitopes in the ZEBRA protein, we synthesized a set of overlapping peptides and tested them for reactivity with serum samples from EBV-seronegative persons, patients with NPC, IM, chronic fatigue syndrome, lymphoma or from healthy donors. Three major EBV-specific epitopes were found. These epitopes were further defined and optimized using substitution or truncation analogues of the peptides. Reactivity with epitope number 22 was found in 63% of NPC patients' sera, with < 2% of healthy donors' sera being positive. Serological reactivity with epitope number 19 was associated with IM (57% positive, 5% healthy donors positive). Serum antibodies against epitope 1 were found among healthy donors, but were significantly elevated among patients with NPC, IM or lymphomas. In conclusion, different serologically reactive epitopes in the ZEBRA protein associate with different EBV-associated diseases.