Epstein-Barr virus (EBV) has been implicated in the pathogenesis of acquired immunodeficiency syndrome- (AIDS) related primary central nervous system (CNS) lymphoma. Tumors from 16 patients with AIDS-related primary CNS lymphoma, and 1 with concurrent CNS and systemic lymphoma, were evaluated histologically and for the presence of EBV by immunohistochemistry for latent membrane protein, in situ hybridization for EBER1 RNA transcripts, polymerase chain reaction (PCR) for the single copy EBNA1 gene, and PCR for the multiple copy EBV internal repeat region. Histologically, 11 tumors displayed extremely large, bizarre, anaplastic immunoblasts, with prominent nucleoli and multilobated nuclei, resembling Reed-Sternberg (RS) cells and variants. The lymphomas displayed B cell phenotypes by immunohistochemistry. Latent membrane protein was detected in 88% (15/17) of tumors, EBNA1 sequences in 54% (6/11), EBV-internal repeat sequences in 100% (11/11), and EBER1 transcripts in 100% (17/17). All EBNA1-negative tumors lacked RS-like cells. Latent membrane protein immunohistochemical staining was limited to a minority of tumor cells, and was most often positive in RS-like, immunoblastic large cells. In situ hybridization for EBER1 message demonstrated EBV in the majority of tumor cells, which displayed a wide range of sizes and variable nuclear morphology. We conclude that EBV can be detected in all AIDS-related primary CNS lymphomas, and that EBER1 in situ hybridization is currently the best technique for detecting virus. The presence of atypical immunoblasts (RS-like cells) with high levels of latent membrane protein in many of these tumors may suggest the emergence of a common, virally-determined phenotype in AIDS-related lymphomas of brain.