The Epstein-Barr virus BamHI F promoter (Fp) was previously identified as the putative EBNA 1 gene promoter in group 1 Burkitt's lymphoma (BL) cell lines. Fp has also been shown to be activated in Epstein-Barr virus-positive B-cell lines following induction of the viral productive cycle (A. L. Lear, M. Rowe, M. G. Kurilla, S. Lee, S. Henderson, E. Kieff, and A. B. Rickinson, J. Virol. 66:7461-7468, 1992). Here we demonstrate that Fp is exclusively a lytic promoter which was incorrectly identified as the EBNA 1 gene promoter in group 1 BL cell lines. It is shown that while Fp activity was observed in two group 1 BL cell lines, it could not be detected in a third group 1 BL cell line. Furthermore, the level of Fp activity detected in both group 1 and group 3 cell lines appeared to correlate only with the level of spontaneous lytic activity. Induction of the lytic cycle in group 1 or group 3 BL cell lines resulted in a dramatic increase in Fp-initiated transcripts but no detectable increase in EBNA 1 transcripts. Anti-immunoglobulin induction of the lytic cycle in the Akata group 1 BL cell line revealed that induction of Fp activity was detectable by 2 to 4 h after induction of the lytic cycle and was dependent on de novo protein synthesis. In addition, Fp reporter constructs transiently transfected into group 1 BL cell lines exhibited activity which was independent of the Fp initiation site, TATAA box, or other upstream sequences. The sequences required for efficient reporter gene activity mapped to a region ca. 210 bp downstream of the Fp cap site. Furthermore, Northern (RNA) blot analyses indicated that there are two Fp-initiated lytic transcripts between 9 and 15 kb in size, neither of which correspond to the known EBNA 1 transcripts present in group 1 BL cell lines.