The Epstein-Barr nuclear antigen-1 (EBNA-1) is a protein containing a large glycine-alanine repeat that has been shown to be antigenic. Antibodies to EBNA-1 can be detected by means of immunoblotting. Preincubation of antisera with purified EBNA-1 protein inhibits the binding of IgG antibodies in this system, indicating that those epitopes detected by immunoblots are also accessible on the native molecule. A number of synthetic peptides the sequences of which were derived from the glycine-alanine repeating region of EBNA-1 and from regions adjacent to it also inhibited antibody binding to EBNA-1. These showed, however, a 1000-fold variation in their inhibitory activities. Peptides containing only glycine and alanine were the most effective inhibitors. The anti-EBNA-1 antibodies did not react with several other peptides representing sequences from unrelated proteins. At saturating concentrations of peptide 85 to 100% of anti-EBNA-1, antibody binding was inhibited in all sera tested with one exception. Similar results are obtained when antibody binding is assayed by an enzyme immunosorbent assay by using partially purified EBNA-1 to coat the plates. Thus the glycine-alanine region, either through its primary structure or through conformations assumed by this region, forms the major epitope(s) of the EBNA-1 molecule.