Expression of the lytic cycle genes of Epstain-Barr virus (EBV) is induced in type I Burkitt's lymphoma-derived cells by treatment with phorbol esters (e.g., phorbol myristate acetate [PMA]), anti-immunoglobulin, or the cytokine transforming growth factor beta (TGF-beta). Concomitantly, all these agents induce apoptosis as judged by a sub-G1 fluorescence-activated cell sorter (FACS) profile, proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. However, caspase activation is not required for induction of the lytic cycle since the latter is not blocked by the caspase inhibitor ZVAD. Furthermore, not all agents that induce apoptosis in these cultures (for example, cisplatin and ceramide) induce the EBV lytic programme. Although it is closely associated with the lytic cycle, apoptosis is neither necessary nor sufficient for its activation. Multiparameter FACS analysis of cultures treated with PMA, anti-Ig, or TGF-beta revealed BZLF1-expressing cells distributed in different phases of the cell cycle according to which inducer was used. However, BZLF1-positive cells did not appear to undergo apoptosis and accumulate with a sub-G1 DNA content, irrespective of the inducer used. This result, which suggests that lytic gene expression is protective, was confirmed and extended by immunofluorescence staining doubled with TUNEL analysis. BZLF1- and also gp350-expressing cells were almost always shown to be negative for TUNEL staining. Similar experiments using EBV-positive and -negative subclones of Akata BL cells carrying an episomal BZLF1 reporter plasmid confirmed that protection from apoptosis was associated with the presence of the EBV genome. Finally, treatment with phosphonoacetic acid or acyclovir prior to induction with PMA, anti-Ig, or TGF-beta blocked the protective effect in Mutu-I cells. These data suggest that a late gene product(s) may be particularly important for protection against caspase activity and cell death.