To precisely determine the genotype of Epstein-Barr virus (EBV) in Hodgkin's disease (HD), we simultaneously analyzed three divergent gene loci (EBNA-2, EBNA-3C, and EBER) that distinguish type A and B viruses. The primers designed to amplify these three gene loci encompass either type-specific deletion sequences (EBNA-2 and EBNA-3C) or type-specific point mutations (EBER) that identify the virus strain based on the sizes of the polymerase chain reaction (PCR)-amplified products or the mobility shifts in single-strand conformation polymorphism analysis. The locations of point mutations were identified by direct sequencing of the PCR-amplified DNA. We analyzed 15 EBV-infected cell lines and found a good correlation between EBNA-2 and EBNA-3C typing results. In contrast, approximately 33% of the cell lines analyzed maintained type A sequences in EBNA-2 and EBNA-3C genes while carrying type B sequences in the EBER region. Data obtained from analysis of cell lines served as a reference for studying HD samples. EBV DNA was detected in about 70% of HD. Among the EBV-positive samples, 56% were associated with type A virus, 13% with type B, and 31% with dual viral sequences. Thus, type A virus is predominant in HD. Based on the histology, the frequencies of EBV positivity were 83%, 71%, and 33% for mixed cellularity, nodular sclerosis, and lymphocyte predominance, respectively. The detection of high frequency of both type A and B sequences in HD may provide a lead in investigating the role of dual viral infection in EBV pathogenesis.