Protein kinase CK2 is an attractive target for anti-neoplastic and antiviral drugs and the inhibitors of CK2 have clinical therapeutic potential. The effect of recombinant human CK2 holoenzyme activity and cellular CK2 activity by digitoflavone was assayed by detecting incorporation of ³²P of [γ-³²P]ATP into the substrate. The mRNA expression of CK2α, α' and β subunits were detected by multiplex RT-PCR. CK2 kinetic analysis was carried out by using the Lineweaver-Burk plot. Digitoflavone was shown to inhibit strongly the activity of recombinant human CK2 holoenzyme in vitro (IC₅₀=0.86 μmol/L) and endogenous CK2 in HL-60 cells, which was more effective than the positive control 4Br-2-azabenzimidazole (TBB). After being treated for 2 h with digitoflavone, the mRNA expression of CK2α and α' subunit decreased, while β subunit was not changed fundamentally. Kinetic studies of digitoflavone on recombinant human CK2 showed that digitoflavone acted as an inhibitor of competitive with ATP and mixed types with casein. These results indicating that digitoflavone is a potent endogenous inhibitor of protein kinase CK2.