The Epstein-Barr virus (EBV) open reading frame BHRF1, a homologue of the oncogene bcl-2, was cloned from a patient with nasopharyngeal carcinoma (NPC) and overexpressed in Escherichia coli. The resulting recombinant BHRF1 fusion protein, with an apparent molecular weight of 35 KD, was used as antigen in an immunoblotting assay for IgG antibody in human sera. Anti-BHRF1 antibody was detected in 57 (61.3%) of 93 patients with NPC, 5 (5.7%) of 87 patients with nonmalignant diseases of the nasopharynx, and in 1 (1.3%) of 78 healthy blood donors. The positivity rate in these nonmalignant patients was 4.4 times that of the normal controls. Negative results were observed in four patients with infectious mononucleosis and patients with other cancers, including 4 with esophageal cancer, 11 with lung cancer, 10 with lymphoma, 13 with gastric carcinoma, 10 with cervical carcinoma, and 10 with other head and neck cancers. Antibody neutralizing EBV DNase and IgA antibody to viral capsid antigen (VCA) were assayed in parallel. The results showed that 7.5% of the NPC patients were negative for anti-DNase and anti-VCA antibodies and EBV infection could be detected by the anti-BHRF1 antibody alone. The demonstration of anti-BHRF1 antibody in most NPC sera strongly supports the hypothesis that the EBV BHRF1 protein is expressed in most NPC patients and its specific antibody can be a useful marker for the diagnosis of NPC.