Five peptides were synthesized on the basis of amino acid sequences predicted from the transformation-associated BamHI WYH region of the genome of the Epstein-Barr virus (EBV). Antisera to two peptides deduced from a 1.6-kb open reading frame in the BamHI H fragment identified an 87 000-dalton nuclear polypeptide that was present in EBV-carrying cell lines that expressed the second EBV-determined nuclear antigen (EBNA-2). This polypeptide was not detected in cell lines that carried EBV variants with a deleted BamHI WYH region or in EBV-negative cell lines. Three peptides deduced from the 1.6-kb open reading frame reacted with human EBNA-positive sera, but not with EBNA-negative sera. Following affinity purification with the peptides, two of the corresponding human antibodies also reacted with the 87 000-dalton polypeptide.