Epstein-Barr virus (EBV) is associated with a number of different human tumors and appears to play different pathogenetic roles in each case. Thus, immunoblastic B cell lymphomas of the immunosuppressed display the full pattern of EBV latent gene expression (expressing Epstein-Barr nuclear antigen [EBNA]1, 2, 3A, 3B, 3C, and -LP, and latent membrane protein [LMP]1, 2A, and 2B), just as do B lymphoblastoid cell lines transformed by the virus in vitro. In contrast, those EBV-associated tumors with a more complex, multistep pathogenesis show more restricted patterns of viral gene expression, limited in Burkitt's lymphoma to EBNA1 only and in nasopharyngeal carcinoma (NPC) to EBNA1 and LMP1, 2A, and 2B. Recent evidence has implicated EBV in the pathogenesis of another lymphoid tumor, Hodgkin's disease (HD), where the malignant Hodgkin's and Reed-Sternberg (HRS) cells are EBV genome positive in up to 50% of cases. Here we extend preliminary results on viral gene expression in HRS cells by adopting polymerase chain reaction-based and in situ hybridization assays capable of detecting specific EBV latent transcripts diagnostic of the different possible forms of EBV latency. We show that the transcriptional program of the virus in HRS cells is similar to that seen in NPC in several respects: (a) selective expression of EBNA1 mRNA from the BamHI F promoter; (b) downregulation of the BamHI C and W promoters and their associated EBNA mRNAs; (c) expression of LMP1 and, in most cases, LMP2A and 2B transcripts; and (d) expression of the "rightward-running" BamHI A transcripts once thought to be unique to NPC. This form of latency, consistently detected in EBV-positive HD irrespective of histological subtype, implies an active role for the virus in the pathogenesis of HD and also suggests that the tumor may remain sensitive to at least certain facets of the EBV-induced cytotoxic T cell response.