Thirty-one cases of Hodgkin's disease were examined for the occurrence of Epstein-Barr virus (EBV) genome by using the polymerase chain reaction (PCR) of DNA in formalin-fixed paraffin-embedded tissues and the in situ hybridization technique. The cases were subdivided into 17 cases of nodular sclerosis (NS), nine cases of mixed cellularity (MC), four cases of lymphocyte predominance (LP), and one case of lymphocyte depletion (LD). EBV DNA was detected in eight cases including four cases of NS, three cases of MC and one case of LP. The sensitivity of PCR was higher than that of Southern blot hybridization of DNA from fresh frozen tissue, because Southern blot hybridization using the BamHI-W fragment of EBV detected virus DNA only in two of three cases which were positive by PCR. The results of in situ hybridization studies confirmed that EBV genome was localized within the nuclei of Reed-Sternberg (RS) cells and their mononuclear variants. Furthermore, double-labeling studies combining in situ hybridization and immunocytochemistry using CD30 (BerH2) and CD15 (LeuM1) as markers of RS cells, as well as pan B-marker (L26) and pan T-marker, CD45RO (UCHL1), were performed to demonstrate the phenotype of EBV DNA-positive cells, confirming that EBV DNA was present in RS cells but not in lymphocytes. The results of this study indicate a significant association between EBV and some cases of Hodgkin's disease.