Epstein-Barr virus (EBV) is a causative agent of nasopharyngeal carcinoma (NPC), and EBV gene expression is considered to be closely associated with the pathogenesis of NPC. Among EBV genes expressed in NPC, EBV-encoded non-polyadenylated RNAs, termed EBERs, are the most abundant transcripts of EBV in NPC. However, the role of EBERs still remains unclear. This study was designed to investigate the relevance of EBERs to the oncogenesis of NPC.Two types of EBERs expression vectors (EBERs-high-expression vector and EBERs-low-expression vector) were constructed and transfected into EBV-negative cells, MDCK or EBV-negative clones of NPC-KT cells. Then, malignant transformation, represented by anchor independent growth, was evaluated between the EBERs-transfected cells and EBERs-negative cells using a soft agar colony formation assay. Apoptosis was induced by serum deprivation (0.1% concentration of fetal bovine serum) and interferon-alpha (IFN-alpha) (500 U/ml) treatment. Cell viability was evaluated with a trypan blue exclusion test. The activation of cellular transcriptional factor NF-kappaB was studied with the IL-8 promoter sequence using a luciferase reporter assay.EBERs-high-expression vector-transfected MDCK cells showed enhanced growth ability in soft agar compared with either EBERs-low-expression vector-transfected MDCK cells or EBERs-untransfected MDCK cells. However, they did not show the acquisition of any anti-apoptotic potential against either IFN-alpha or serum deprivation. Introduction of EBERs-low-expression vector into MDCK cells did not show anchor independent growth characteristics. Neither EBV-negative NPC-KT cells nor MDCK cells transfected with EBERs-high-expression vector showed any difference from EBERs-untransfected EBV-negative NPC-KT cells. Introduction of EBERs into MDCK cells did not transactivate the IL-8 promoter, indicating that neither NF-kappaB nor AP-1 was activated by EBERs.EBERs are believed to induce the initial transformation of epithelial cells, thus contributing to the oncogenesis of NPC. Expression of abundant EBERs is considered to be critical for this transforming property of EBERs.