To elucidate the association of Epstein-Barr virus (EBV) and salivary gland tumors, 114 cases of tumors of major salivary glands were investigated. EBV DNA was detected in all 6 cases of undifferentiated carcinoma and all 3 cases of T-cell lymphoma, but not in other tumor tissues. In situ hybridization studies for EBV DNA and EBV-encoded small RNA 1 (EBER1) showed specific localization of the EBV sequences to the undifferentiated carcinoma cells and T-lymphoma cells. Moreover, intense DNA signals were detected on nonneoplastic epithelial cells of T-lymphoma tissues. These epithelial cells were negative for EBER1 and expressed BZLF1, BALF2, and gp350/220 proteins associated with virus production. In contrast, nonmalignant epithelial cells surrounded by undifferentiated carcinoma cells showed no evidence of EBV infection or virus replication. These results indicate that there is an unusual association of salivary gland T-cell lymphomas with lytic EBV replication of nonmalignant epithelial cells.