The EBV nuclear antigen, EBNA1, is the only viral protein consistently expressed in all virus-infected cells. It is required in trans for viral replication, maintenance of EBV extrachromosomal episomes, and transcriptional transactivation in latently-infected B-cells. It binds RNA suggestive of a regulatory role in post-transcriptional events and in transgenic mice, it is tumorigenic. In RNase protection studies relating to the EBV-associated tumour, nasopharyngeal carcinoma (NPC), we show that a C-terminal EBNA1 RNA probe from the prototype B95-8 marmoset strain can protect its own mRNA from enzymatic digestion, but does not fully protect EBNA1 mRNA from NPC cells. This finding is consistent with changes in the coding region for the antigen. We thus determined the sequences of EBNA1 genes derived from an NPC xenograft and numerous patient biopsies and identified a number of mutations in the gene in these human cells, relative to B95-8. Many of the nucleotide changes would lead to non-conservative amino acid alterations in apparently functionally significant regions of the protein. We show that although some of the mutations lie in regions designated as critical to DNA binding, they have negligible effect on this property of EBNA1. The basic regions in EBNA1 that may bind to RNA, at least in vitro, are exempt from mutation. Thus, unless the alterations are 'silent', which for such a critical viral function seems unlikely, they may relate to as yet unmapped viral activities, such as a role in tumorigenesis and the ability of EBNA1 to evade the cellular immune system, or be associated with the ability of the antigen to regulate gene transcription.