The Epstein-Barr virus (EBV) nuclear antigen EBNA-1 is essential for viral genome maintenance in vitro and may be the only EBV protein expressed by the majority of latently infected cells in vivo. EBNA-1 may therefore be critical to the evasion of host immunity which allows persistent infection. EBNA-1 includes a polymorphic internal repeat domain of unknown significance and unique regions which mediate all known functional activities and which have hitherto been assumed to be conserved between strains. Monoclonal antibodies were generated using a construct based on EBNA-1 of the prototype B95-8 strain, deleted for the repeat domain. These antibodies showed a limited profile of recognition of EBNA-1 in common laboratory EBV+ cell lines by immunoprecipitation and immunostaining. The observed antigenic heterogeneity also extended to spontaneously transformed B lymphoblastoid cell lines (LCLs) representing viral isolates circulating within US and UK populations. DNA fragments spanning the C-terminal unique domain of EBNA-1 from eleven spontaneous LCLs were amplified by polymerase chain reaction for sequencing, which directly demonstrated extensive and unexpected variability between diverse type 1 EBV isolates. The resulting polymorphism affects most of the putative MHC Class I binding epitopes which could be identified within this region using published sequence motifs, and influences MHC binding by variants of at least one such peptide in the processing mutant cell line T2. These findings could be related to the apparent lack of recognition of EBNA-1 by cytotoxic T lymphocytes.