Epstein-Barr virus (EBV) gene expression in nasopharyngeal carcinoma (NPC) includes abundant rightward transcription of the BamHI A fragment, consisting of mRNAs ranging in size from approximately 4.0 to 8.0 kb. These transcripts include several distinctly spliced forms which are 3'-end coterminal and contain the BamHI A rightward frame 0 (BARF0) open reading frame (ORF) in the final exon. BamHI A transcription is detected at a lower level of expression in EBV-infected lymphoid cells. In this study, cDNA cloning, reverse transcription-based PCR, and Northern (RNA) blotting were used to further define the structures of the BamHI A transcripts and to characterize their expression in different EBV-infected tissues. Three BamHI A cDNAs isolated from a passaged NPC represent previously unidentified mRNAs that contain BARF0 and additional ORFs encoded by multiple exons, including one which extends the size of the BARF0 ORF from 174 to 279 codons. The distinct exons were detected in multiple, differently sized mRNAs, indicating that these transcripts have complex patterns of alternate splicing. In support of this finding, 5'-end analysis confirmed the presence of a previously reported start site and also identified a subset of transcripts of 4.8 kb and larger that initiate further 5' to this site. In addition, 3'-end analysis identified heterogeneous 3'-end processing in all of the BamHI A mRNAs, resulting in transcripts that either contain the entire BARF0 ORF or are cleaved and polyadenylated 5' of the stop codon. Finally, the expression of multiple, distinctly spliced BamHI A transcripts was consistently detected in a wide range of EBV-infected samples, including NPC, Burkitt's lymphoma, and parotid carcinoma biopsy samples, and in type I and type III Burkitt's lymphoma lines and type III lymphoblastoid cell lines. This complex pattern of start site selection, alternate splicing, and heterogeneous 3'-end processing is likely to regulate the expression in vivo of the ORFs encoded by the EBV BamHI A transcripts.