Several cDNA clones containing the Epstein-Barr virus BamHI fragment H rightward open reading frame 1 (BHRF1) have been recovered from the tightly latent lymphoblastoid cell line IB4. These clones contain the 5' leader exons encoded in the major internal repeat 1 and the viral BamHI fragment Y, identified in the rightwardly transcribed viral mRNAs associated with the latent viral life cycle. In addition, a cDNA clone containing BHRF1 from the Burkitt lymphoma cell line Jijoye was also recovered and exhibits a distinctive splicing pattern. In vitro transcription and translation of BHRF1, followed by immunoprecipitation with Epstein-Barr virus-positive human sera, indicates that this viral antigen is expressed during infection. RNA blot analyses with a wide panel of lymphoblastoid and Burkitt lymphoma cell lines revealed a complex pattern of transcription. Hybridization data obtained with several probes is presented.