In situ hybridization, using a biotinylated sequence from the internal repeat (IR1) region of Epstein-Barr virus (EBV), was performed on two well characterized EBV-infected cell lines, B95-8 (productively infected) and Namalwa (latently infected), and on eighteen formalin-fixed paraffin-embedded primary central nervous system (CNS) lymphomas. Ten of the lymphomas were from immunocompetent patients, and eight were from immunocompromised patients (five had acquired immunodeficiency syndrome (AIDS), two had renal allografts, and one had X-linked immunoproliferative (XLP) disease). Both fresh and paraffin-embedded B95-8 cells showed detectable hybridization signal in 5 to 10% of cells, with other cells showing lower signal. Fresh Namalwa cells showed signal in every cell and in 40% of paraffin-embedded cells. Evidence of EBV genome was seen in seven of eight lymphomas from immunocompromised patients and in none of the lymphomas from immunocompetent ones. In the EBV-positive lymphomas, three patterns of hybridization were recognized: +3, more than 60% of tumor cells positive, +2, 20 to 60% of tumor cells positive, and +1, less than 20% of tumor cells positive. There was no definite relationship between survival after diagnosis and hybridization pattern type. While the signal in Namalwa cells was uniform, a wide variation in the degree and intensity of signal was noted among the seven positive tumors and even in different areas of the same tumor. This heterogeneity raises the possibility of lytic or secondary infection in a small number of the latently infected tumor cells.