Abstract Phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae catalyzes the formation of phosphatidylinositol 4-phosphate and ADP from phosphatidylinositol and ATP. Phosphatidylinositol 4-kinase catalyzes the first phosphorylation reaction in the reaction sequence phosphatidylinositol → phosphatidylinositol 4-phosphate → phosphatidylinositol 4,5-bisphosphate. This phosphorylation sequence in S. cerevisiae is regulated by glucose and sterol. Phosphatidylinositol 4,5-bisphosphate appears to play an essential role in cell proliferation of S. cerevisiae. Since phosphatidylinositol 4-kinase catalyzes the first step in the phosphorylation sequence of phosphatidylinositol, the enzyme should play a major role in phosphoinositide synthesis and cell growth in S. cerevisiae. Two membrane-associated (45 kDa and 55 kDa) forms and one cytosolic-associated (125 kDa) form of phosphatidylinositol 4-kinase have been purified and characterized from S. cerevisiae. The membrane-associated phosphatidylinositol 4-kinases differ with respect to their physiochemical, enzymological, and kinetic properties. The binding and catalytic steps of the reactions catalyzed by the membrane-associated phosphatidylinositol 4-kinases toward phosphatidylinositol has been been defined through meaningful kinetic analyses using Triton X-100/PI-mixed micelles. Detailed kinetic analyses of the inhibition of membrane-associated phosphatidylinositol 4-kinases by nucleotides has led to insights on the regulation of phosphoinositide synthesis in response to glucose.