Epstein-Barr virus (EBV) genome is associated with a variety of lymphoid and epithelial malignancies. EBV DNA has been detected in some cases of thymic carcinoma, but the cellular locus of the virus has never been defined. Detection of EBV has also been reported in normal thymus, thymic lymphoid hyperplasia, and thymoma by some investigators but not by others. In order to better define the association of the virus with benign and malignant thymic tissues and to characterize its cellular locus, we applied a recently developed in situ hybridization technique using a very abundant EBV transcript (EBER1) as target to a variety of thymic tissues. We detected expression of this transcript only in the malignant epithelial cells in one case of thymic lymphoepithelioma-like carcinoma. EBV expression was not detected in six other cases of thymic carcinoma, nor in tissue from 16 normal thymuses, 14 thymomas, and 10 thymic lymphoid hyperplasias.