The Epstein-Barr virus (EBV) immediate-early proteins BZLF1 and BRLF1 can both induce lytic EBV reactivation when overexpressed in latently infected cells. Although EBV genome methylation is required for BZLF1-mediated activation of lytic gene expression, the effect of viral genome methylation on BRLF1-mediated viral reactivation has not been well studied. Here, we have compared the effect of viral DNA methylation on BZLF1- versus BRLF1-mediated activation of lytic EBV gene transcription and viral genome replication. We show that most early lytic viral promoters are preferentially activated by BZLF1 in the methylated form, while methylation decreases the ability of BRLF1 to activate most early lytic promoters, as well as the BLRF2 late viral promoter. Moreover, methylation of bacmid constructs containing the EBV genome enhances BZLF1-mediated, but decreases BRLF1-mediated, early lytic gene expression. Methylation of viral promoter DNA does not affect BRLF1 binding to a variety of different CpG-containing BRLF1 binding motifs (RREs) in vitro or in vivo. However, BRLF1 preferentially induces H3K9 histone acetylation of unmethylated promoters in vivo. The methylated and unmethylated forms of an oriLyt-containing plasmid replicate with similar efficiency when transfected into EBV-positive cells that express the essential viral replication proteins in trans. Most importantly, we demonstrate that lytic viral gene expression and replication can be induced by BRLF1, but not BZLF1, expression in an EBV-positive telomerase-immortalized epithelial cell line (NOKs-Akata) in which lytic viral gene promoters remain largely unmethylated. These results suggest that the unmethylated form of the EBV genome can undergo viral reactivation and replication in a BRLF1-dependent manner.