A membrane-bound phosphatidylinositol 4-kinase (PI 4-kinase) was separated in a sucrose gradient and solubilized with 1% Triton X-100 from mouse brain. The enzyme was purified 2,952-fold by various chromatographic techniques including DEAF-cellulose, PI-Sepharose and Sephacryl S-200 gel filtration. The molecular weight of PI 4-kinase was approximately 76 kDa by gel filtration and 70.8 kDa by SDS-polyacrylamide gel electrophoresis. The purified enzyme exhibited specific activity of 112 nmol/min/㎎ protein and pI value of 4.7. Kinetic analysis of the PI 4-kinase indicated apparent K_m values of 190 μM and 120 μM for phosphatidylinositol and ATP, respectively. The maximal activity of this purified enzyme was observed at pH 7.4 at an incubation temperature of 37℃. The enzyme activity was significantly activated by Mg^(2+), Mn^(2+) and Fe^(2+), and inhibited severely by Ca^(2+). PI 4-kinase was proved to be pure in its immunoblot test by polyclonal antibody prepared from immunized rabbit sera. By this test, we were able to detect the existence of the same type of PI 4-kinase from other mouse organ tissues, such as liver, heart, kidney and spleen. Furthermore, similar immunoblot analysis with the same antisera recognized the different epitopes of PI 4-kinase proteins from various organs of rabbit, Chinese hamster and rat.