A membrane-bound phosphatidylinositol 4-kinase(PI4K) has been purified approximately 11 500-fold from bovine cerebella cortex. The purification procedure involves: solubilisation of the membrane fraction with TritonX-100, ammonium sulfate fractionation and cation-exchanger chromatography on phosphocellulose followed by affinity chromatography on heparin-sepharose CL-6B. The enzyme was further purified through DEAE-10 FPLC chromatography at last. The purified enzyme exhibited a final specific activity of 450 nmol/mg·min. The molecular mass of the enzyme was estimated to be 56 ku by SDS-PAGE. Kinetic measurements showed that the apparent K_m value of PI kinase for the utilization of PtdIns is 6.6×10^-7 mol/L and for ATP 7.9×10^-7 mol/L. In addition adenosine was found to be a strong inhibitor, Enzymatic activity was found to be stimulated by Triton X-100.