Translation elongation factor 1δ (EF-1δ) is hyperphosphorylated in various mammalian cells infected with alpha-, beta- and gammaherpesviruses and EF-1δ modification is mediated by viral protein kinases, including UL13 of herpes simplex virus type 1 and UL97 of human cytomegalovirus. In this study, the following is reported. (i) BGLF4 encoded by the prototype gammaherpesvirus Epstein-Barr virus was purified as a fusion protein that was labelled with [γ-32P]ATP and labelling was eliminated by phosphatase. (ii) The ratio of the hyperphosphorylated form of human EF-1δ was increased both in Sf9 cells after infection with baculoviruses expressing GST-BGLF4 fusion proteins and in COS-7 cells after transfection with a BGLF4 expression plasmid. These results indicate that purified BGLF4 possesses protein kinase activity and mediates EF-1δ hyperphosphorylation. These data also support the hypothesis that the protein kinases that are conserved by herpesviruses universally mediate EF-1δ modification in mammalian cells.