The BZLF1 or zta immediate-early gene of Epstein-Barr virus (EBV) encodes a 33-kilodalton phosphorylated nuclear protein that is a specific transcriptional activator of the EBV lytic cycle when introduced into latently infected B lymphocytes. We have shown previously that the divergent EBV DSL target promoter contains two zta-response regions, one within the minimal promoter and the other in an upstream lymphocyte-dependent enhancer region. In this study, we used footprinting and gel mobility retardation assays to reveal that bacterially synthesized Zta fusion proteins bound directly to six TGTGCAA-like motifs within DSL. Four of the Zta-binding sites lay adjacent to cellular TATA and CAAT factor-binding sites within the minimal promoter, and two mapped within the enhancer region. Single-copy oligonucleotides containing these Zta-binding sites conferred Zta responsiveness to heterologous promoters. In addition, the Zta protein, which possesses a similar basic domain to the conserved DNA-binding region of the c-Fos, c-Jun, GCN4, and CREB protein family, proved to bind directly to the consensus AP-1 site in the collagenase 12-O-tetradecanoylphorbol-13-acetate response element. Cotransfection with zta also trans activated a target reporter gene containing inserted wild-type 12-O-tetradecanoylphorbol-13-acetate response element oligonucleotides. Cellular AP-1 binding activity proved to be low in latently EBV-infected Raji cells but was induced (together with the Zta protein) after activation of the lytic cycle with 12-O-tetradecanoylphorbol-13-acetate. We conclude that EBV may have captured and modified a cellular gene encoding a c-jun-like DNA-binding protein during its evolutionary divergence from other herpesviruses and that this protein is used to specifically redirect transcriptional activity toward expression of EBV lytic-cycle genes in infected cells.