The BGLF4 protein of Epstein-Barr virus (EBV) is a serine/threonine protein kinase that phosphorylates several viral and cellular substrates at cellular cyclin-dependent kinase target sites. BGLF4 is required for efficient viral DNA replication and release of mature virions. It also stimulates the transactivation activity of the immediate-early transactivator Zta (BZLF1) and suppresses the transactivation activities of BMRF1 and EBNA-2. This study aimed to characterize further the regulation of BGLF4 expression at the transcriptional and translational levels. It was shown that BGLF4 was expressed with early kinetics and reached maximal levels after DNA replication. The promoter activity of BGLF4 was upregulated mainly by the immediate-early transactivator Rta, rather than Zta, as revealed by Zta-specific short hairpin RNA in EBV-positive cells and by luciferase reporter assays. By rapid amplification of 5' cDNA ends, two major transcriptional start sites were identified at 201 and 255 nt upstream of the first in-frame ATG of BGLF4 in P3HR1 cells. An additional transcript initiated from -468 was detected in Akata cells. The translation initiation site of BGLF4 was confirmed by mutagenesis, in vitro translation and transient transfection. The translation regulatory effect mediated by the long 5'-untranslated region (5'UTR) of BGLF4 was demonstrated by dual reporter assays in 293T and EBV-positive NA cells. These results suggested that different promoter usage and 5'UTR-mediated translation enhancement may ensure the proper expression of BGLF4 at various stages of virus replication.