The association of Epstein-Barr virus (EBV) with nasopharyngeal carcinoma (NPC) has been shown by various methods. The purpose of this study is to identify the most useful method to detect EBV in NPC. Both polymerase chain reaction (PCR) for EBV-DNA and in situ hybridization for EBV-encoded small RNAs (EBERs) were examined in formalin-fixed, paraffin-embedded NPC specimens. In situ hybridization was performed in 56 cases, and PCR for EBV-DNA was performed in 42 cases. EBV-DNA was detected in 0 of 3 keratinizing squamous cell carcinomas (KSCC), 22 of 24 nonkeratinizing carcinomas (NKC), all 13 undifferentiated carcinomas (UNPC), and 0 of 2 adenocarcinomas (AC). EBERs were detected in 0 of 5 KSCC, 30 of 32 NKC, 16 of 17 UNPC, and 0 of 2 AC. Among them, EBERs was detected in 35 of 42 cases in which PCR was also performed, 0 of 3 KSCC, 22 of 24 NKC, all 13 UNPC, and 0 of 2 AC, respectively. Both results were consistent in 40 of 42 cases. We conclude that both PCR and in situ hybridization are useful to detect EBV in NPC. In situ hybridization has a particular advantage because it can demonstrate the localization of EBV in neoplastic cells. In addition, close association of NKC and UNPC but not KSCC and AC with EBV is suggested.