We have established a cell line (C666‐1) from undifferentiated nasopharyngeal carcinoma (NPC). This cell line consistently carries the Epstein‐Barr virus (EBV) in long‐term cultures. C666‐1 is a subclone of its parental cell line, C666, derived from an NPC xenograft of southern Chinese origin. It grows as an adherent culture and lacks contact inhibition. In addition, it is tumorigenic in athymic nude mice. The cells consistently express EBV‐encoded RNAs and are positively stained for cytokeratin, an epithelial marker. In addition, they express EBNA1 protein, LMP1 and LMP2 transcripts and thus resemble the EBV latency II pattern. The virus genotype is EBV‐1 with the latent membrane protein 1 gene showing a 30‐bp deletion at the carboxyl terminus, both consistent with findings in southern Chinese NPC tumours. Cytogenetic analysis revealed a sub‐diploid status with a chromosomal modal number of 45. C666‐1 is unique among NPC cell lines in that it carries EBV. These cells may serve as a good investigative tool as the viral latency pattern and genotype are observed in the majority of primary NPC biopsies from Chinese patients. Int. J. Cancer 83:121–126, 1999. © 1999 Wiley‐Liss, Inc.