Primary non-Hodgkin S lymphoma of the central nervous system (PCNSL) has recently increased in incidence, due primarily to an enlarging immunosuppressed patient population. The pathogenetic role of Epstein-Barr virus (EBV) is of interest due to its established role in other lymphoproliferative disorders in immunosuppressed patients. Twenty-three cases of histologically confirmed PCNSL with corresponding cytology were identi$ed, all obtained under stereotactic guidance. Twenty patients were human immunodeficiency virus (HIV) positive, two were HIV negative, and one was of unknown status. Papanicolaou-stained slides were selected from each case and evaluated for the presence of EBV RNA via in situ hybridization (ISH) utilizing a biotinylated probe specific for EBER I RNA, and detected by a conventional streptavidin-peroxidase system. The cases included immunoblastic (IZ), large cell (IO). and mixed small and large cell lymphoma (I). The predominant immunophenotype was B-cell (19), although T-cell (2) and biphenotypic ( I ) cases were also identij7ed. ISH showed nuclear positivity for EBV RNA in I 9 of 23 cases (83%). This study confrms the presence of EBV in PCNSL in immunosuppressed patients and implies a potential etiologic role. The ability to demonstrate EBV RNA in cytologic preparations by ISH also raises the possibility of early identification of high-risk patients through detection of EBV-infected lymphocytes in CSF specimens. Diagn Cytopathol 1996;14:114-120.