The Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC) and with lymphoepithelioma-like carcinomas developing in certain anatomic sites. In this study, an in situ hybridization was used to identify EBV-encoded ribonucleic acid (RNA) (EBER1) transcripts in 32 of 45 cases of NPC but not in any of the 11 lymphoepithelioma-like carcinomas developing in the urinary bladder. EBER1 was most commonly detected in those NPCs having undifferentiated or nonkeratinizing squamous histology rather than the keratinizing squamous cell subtype of NPC. The EBV-encoded latent membrane protein 1 (LMP1) was expressed focally in only seven of 21 EBER1-positive NPCs by an immunohistochemical technique. These findings imply that EBER1 hybridization is more sensitive than LMP1 immunohistochemistry on paraffin sections in detecting carcinoma-associated virus. Previous in vitro studies have suggested that LMP1 expression might be a function of differentiation, but this study of naturally infected NPCs showed no strong correlation between LMP1 positivity and degree of tumor differentiation, albeit a limited spectrum of differentiation that could be examined. In two cases in which frozen tissue was available, the NPCs were monoclonal with respect to viral DNA structure, implying that the virus was present before malignant transformation. Unlike NPCs, the lymphoepithelioma-like carcinomas of the bladder were uniformly EBV negative, lending further evidence to the growing body of literature linking EBV with lymphoepithelial carcinomas of foregut-derived tissues but not with similar-appearing tumors developing in other anatomic sites.