The Epstein-Barr virus (EBV) has been implicated in the pathogenesis of Hodgkin's disease, with an frequency of 15 to 50% in the immunocompetent host. We studied 12 formalin-fixed, paraffin-embedded cases of Hodgkin's disease occurring in human immunodeficiency virus-infected individuals to determine the frequency of EBV in Hodgkin's disease from this population. EBV DNA-RNA in situ hybridization was performed using a 30-base biotinylated anti-sense oligonucleotide complementary to the EBER1 gene of EBV. EBV RNA was found in the Reed-Sternberg cells and variants in 11 of 12 cases. Double-labeling studies confirmed the presence of EBV RNA in CD15-expressing Hodgkin's cells in all 11 cases, although rare B lymphocytes coexpressing EBV RNA and CD20 were also noted in these cases. The Hodgkin's cells in all 11 EBER-positive cases expressed latent membrane protein. The one case negative for EBV RNA showed the histology of nodular, lymphocyte predominance, a subtype thought to be distinct from other types of Hodgkin's disease.