Four promoters, Cp, Wp, Fp and Qp, are known to participate in transcription of the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) gene in EBV-infected cell lines. The promoters are used differentially during the different phases of infection and establishment of the stages of latency. This has raised questions about the regulation of the promoters and the molecular mechanisms underlying the switches between them. To obtain a measure of the activity of the different EBNA1 transcription units in EBV-transformed cell lines of different phenotypes, RNA probes were constructed that allowed the detection and relative quantification, by RNase protection analysis, of EBNA1 transcripts initiated at Fp and Qp and, in an indirect manner, Cp/Wp. RNase protection and PCR assays were performed with cytoplasmic RNA from B-lymphoid cell lines in latency stages I, II-III and III and after induction of the virus lytic cycle. The experiments demonstrated that, in addition to previously identified EBNA1 transcripts, cell lines of all latency types also contained different mRNAs that carried sequences from the EBNA1-encoding K exon. Induction of the virus lytic cycle resulted in low levels of an FpQ/U/K-spliced transcript. However, there was a large increase of FpQ- and FpQ/U-spliced transcripts with unknown 3' sequences. Furthermore, a new transcript, initiated at an unidentified site 5' of the BamHI f/K cleavage site and continuing through BamHI K into the EBNA1-encoding K exon without interruption, was produced in substantial amounts in the lytic cycle.