Transcription of Epstein-Barr virus (EBV) genes in epithelial tissue, one of the two principal cell types infected by EBV, is not well characterized. EBV transcription in a nasopharyngeal carcinoma established in nude mice, C15, has been analyzed by using strand-specific RNA probes and sequence analysis of a C15 cDNA library. In C15, two equally abundant mRNAs of 3.7 and 2.8 kilobases (kb) are encoded by the sequences that encode latent membrane protein (LMP). Hybridization with probes specific for the 3' end of the LMP mRNA to Northern (RNA) blots and sequence analysis of cDNAs representing the messages indicated that the 3.7- and 2.8-kb mRNAs are 3' coterminal. Sequence analysis of additional cDNAs revealed an mRNA that is spliced identically to the LMP mRNA but is initiated 5' to the promoter for LMP. A probe representing the sequences contained within the cDNA which are 5' to the LMP promoter identified the 3.7-kb mRNA in C15 and a low-abundance 3.7-kb mRNA in B95-8 RNA. These data indicate that transcription of the LMP-encoding sequences is complex and that LMP can be expressed from an additional RNA in both nasopharyngeal carcinoma and lymphoid cells. Hybridization with BamHI-A identified a predominant 4.8-kb mRNA and two less abundant larger-molecular-weight mRNAs transcribed in C15. These mRNAs are consistently expressed in all passages in nude mice of the C15 tumor. Hybridization with strand-specific probes and sequence analysis of three cDNAs revealed that these mRNAs are transcribed from left to right. Sequence analysis of cDNAs representing the 3' end of the mRNAs identified an open reading frame that could potentially encode a protein of 174 amino acids. In situ hybridization of a 35S-labeled RNA probe homologous to the BamHI-A cDNA to tissue sections revealed that the BamHI-A mRNA is not focally expressed and is transcribed in all cells within the C15 tumor. Linear forms of EBV DNA were not detected in any of the C15 tumors, and replicative viral antigens have not been detected. These data suggest that the C15 tumor represents a latently infected tumor and that the transcription from BamHI-A, which is expressed in all cells, is not associated with virus replication.