The BZLF1 gene controls the switch between latent and lytic infection by Epstein-Barr virus (EBV). We previously reported that both the ZV and ZIIR elements within the BZLF1 promoter, Zp, are potent transcription silencers within the context of an intact EBV genome. We report here identification of another sequence element, ZV', which synergized with ZV in repressing Zp via binding ZEB1 or ZEB2. We then determined the phenotype of a variant of EBV strain B95.8 in which the ZV, ZV', and ZIIR elements were concurrently mutated. HEK293 cell lines infected with this triple mutant (tmt) virus spontaneously synthesized 6- to 10-fold more viral BZLF1, BRLF1, BMRF1, and BLLF1 RNAs, 3- to 6-fold more viral Zta, Rta, and EAD proteins, 3- to 5-fold more viral DNA, and 7- to 9-fold more infectious virus than did 293 cell lines latently infected with either the ZV ZV' double mutant (dmt) or ZIIR mutant (mt) virus. While ZV ZV' ZIIR tmt EBV efficiently infected human primary blood B cells in vitro, it was highly defective in immortalizing them. Instead of the nearly complete silencing of BZLF1 gene expression that occurs within 4 days after primary infection with wild-type EBV, the ZV ZV' ZIIR tmt-infected cells continued to synthesize BZLF1 RNA, with 90% of them dying within 9 days postinfection. BL41 cells infected with this "superlytic" virus also exhibited increased synthesis of BZLF1 and BMRF1 RNAs. Thus, we conclude that the ZV, ZV', and ZIIR silencing elements act synergistically to repress transcription from Zp, thereby tightly controlling BZLF1 gene expression, which is crucial for establishing and maintaining EBV latency.