Two epithelial tumor cell lines were established from biopsy specimens of 2 nasopharyngeal carcinomas (NPC) and designated HNE-1 and HONE-1. Uncloned HNE-1 cells were found to be Epstein-Barr virus (EBV) DNA-positive when examined by Southern blot analysis up to passage 35, after which the EBV genome could no longer be detected. A similar loss of EBV DNA took place in uncloned HONE-1 cells. However, HONE-1 clone 40 cells are still EBV DNA-positive up to passage 42 thus far and cell cultures contain 85-90% EBV nuclear antigen (EBNA)-positive cells. The HNE-1 cell line has been passaged more than 100 times and the uncloned HONE-1 cells more than 90 times. The tumorigenicity of the HNE-1 and HONE-1 cells was demonstrated by tumor induction in nude mice. Karyotypic analysis of the HNE-1 cells demonstrated an aneuploidy with a modal chromosomal number of 74 at passages 5 and 101 at passage 20; 18 marker chromosomes were identified. We have continued to map the EBV genome latently associated with the HNE-1 and HONE-1 cells using the Bam HI, EcoRI or Hind III restriction enzymes. Using EcoRI fragments A-K as probes, we found that HNE-1 EBV DNA is different from B95-8 and HR-1 EBV DNA in the EcoRI-C region. The Bam HI map for HONE-1 EBV DNA is very similar to the B95-8 map; it contains the Bam HI-Y fragment but without Bam HI B' and WI'. Differences were observed between HONE-1 EBV DNA and B95-8 DNA using the Hind III restriction enzyme. There was no evidence of spontaneous expression of the latent EBV genome in HNE-1 cells, and attempts to induce replication of the latent EBV genome and rescue infectious virus have failed, suggesting a tightly restricted virus genome.