Different activation of Epstein-Barr virus (EBV) promoters results in distinct expression patterns of EBV nuclear antigens (EBNAs) and may further decide the role of EBV in the cellular pathogenesis. In EBV-associated nasopharyngeal carcinoma (NPC) biopsies, it has generally been believed that Q promoter (Qp)-initiated EBNA1 is the only EBNA gene to be expressed and that the other two viral promoters, Cp and Wp, which can lead to expression of EBNA1-6, are inactive. However, the failure to demonstrate the activities of Cp and Wp may have been due to the limited sensitivities of detection approaches used. In the present article, the EBV promoter usage and gene expression were re-examined in both EBV-infected NPC cells in vitro and NPC biopsies in vivo. An NPC cell line susceptible to EBV infection in vitro was established by transfection with a plasmid expressing a well-known EBV receptor, CR2. The presence of viral DNA and EBNA proteins was demonstrated in these EBV-infected cells using PCR and anticomplement immunofluorescence assay, respectively. As has been identified in NPC biopsies, viral transcripts of Qp-initiated EBNA1, latent membrane protein (LMP)1, LMP2A, LMP2B, and BamHI A genes, as well as the EBV-encoded small RNA (EBER)1 were detected in these in vitro-infected cells using reverse-transcription-PCR. Notably, viral transcripts initiated from Cp or Wp were also found in the infected cells. Furthermore, Cp- or Wp-initiated transcripts and EBNA2 mRNA were detected in some NPC biopsies. Taking advantage of this sensitive detection approach, our observation that Cp and Wp may be active in NPC cells raises the possibility that EBNA2 to 6, in addition to EBNA1, may play roles in the pathogenesis of NPC.