Gene mapping of an Epstein-Barr virus (EBV) isolate derived from a nasopharyngeal carcinoma (NPC), designated NPC-EBV, has been performed. This isolate was rescued from an NPC epithelial hybrid cell line (NPC-KT), and used to transform cotton-top tamarin lymphocytes which, along with the epithelial NPC-KT cells, were used in the mapping studies. Using the BamHI and EcoRI restriction enzymes, we found that the NPC-EBV isolate did not contain the deletions observed in the genomes of the prototype HR-1 and B95-8 isolates, and may represent a 'more complete' virus genome. Polymorphism was observed in the BamHI-X, T, H, and L regions as compared to the HR-1 and B95-8 isolates. The NPC-EBV DNA in the epithelial NPC-KT cells was compared to the NPC-EBV genome in three different cotton-top tamarin lymphoblastoid cell lines transformed with the isolate. The restriction patterns were the same with one exception; there were differences in the size of the BamHI N-J het regions. This finding is consistent with the idea that the BamHI N-J het region may be the switch point of EBV DNA from the linear to the episomal form. The NPC-EBV is the first NPC-derived isolate to be obtained from epithelial cells in vivo and maintained in epithelial cells in vitro, and will be useful for studying biological variability of EBV.