BACKGROUND The Epstein-Barr virus (EBV) has been frequently detected in lymphoid malignancies. However, EBV infection in the nonneoplastic cells of lymphoid malignancies has not been extensively studied. METHODS Four hundred nine cases of lymphoid malignancies including 377 non-Hodgkin's lymphoma (NHL) and 32 Hodgkin's disease (HD) were examined for EBV infection by EBER-1 in situ hybridization (EBER-ISH), immunostaining against LMP-1, Epstein-Barr nuclear antigen 2 (EBNA2) and ZEBRA, and Southern hybridization using a BamHIW fragment as a probe. Double staining with EBER-ISH and immunostaining against CD20, CD45RO, and LMP-1 was performed in selected cases. RESULTS Although EBER-1-positive cells (EPCs) were detected in 49 of 276 B-cell lymphomas, 31 of 100 T-cell lymphomas, 1 of 1 natural killer-cell lymphoma, and 17 of 32 HDs, almost all of the tumor cells were exclusively EBER-1-positive in the 10 NHL cases. Some EPCs were of different cell lineages than the tumor cells in 15 of the 26 NHLs examined by double staining. LMP-1, EBNA2, and ZEBRA were detected in 22, 4, and 3 cases, respectively. In 4 LMP-1-positive HDs, double staining revealed that some EBER-1-positive Reed-Sternberg cells were negative for LMP-1, EBV genomic DNA was detected in 8 of the 39 examined cases. CONCLUSIONS T-cell lymphomas contained EPCs more frequently than B-cell lymphomas. Nonneoplastic lymphocytes were infected with EBV more frequently than lymphoma cells. Rowe's latency II may be unstable in lymphoid malignancies. Some NHLs, especially T-cell lymphoma, may provide favorable conditions for EBV infection of nonneoplastic lymphocytes.