The Epstein-Barr virus (EBV) has been implicated in the pathogenesis of Hodgkin's disease (HD). This study was undertaken to determine whether the association of EBV with HD showed geographical variation, as in Burkitt's lymphoma. We studied 32 formalin-fixed, paraffin-embedded cases of HD occurring in Peru. EBV DNA-RNA in situ hybridization was performed using a 30-base biotinylated antisense oligonucleotide complementary to the EBER1 gene of EBV. EBV immunohistochemistry was also performed, using a monoclonal antibody (MoAb) to the latent membrane protein (LMP1) of EBV. Identification of the precise cellular subset staining with EBV was accomplished via double- labeling with MoAbs directed against Reed-Sternberg cells (LeuM1/CD15) and B cells (L26/CD20). EBV RNA was identified in all or virtually all of the Reed- Sternberg cells and variants in 30 of the 32 (94%) cases of HD by in situ hybridization. LMP1 expression was identified in 83% of the EBER1-positive cases. Double-labeling studies confirmed the localization of EBV RNA to CD15- expressing Hodgkin's cells. This study found an extremely high prevalence of EBV in Peruvian HD, in contrast to the much lower percentage of EBV- associated cases of HD occurring in 'Western' patients.