A wide spectrum of intracellular signaling events mediated by up to seven different phosphorylated forms of phosphatidylinositol (PtdIns) occurs in all eukaryotic cells. The activities of multiple, nondegenerate PI kinases and phosphatases control these signaling events. The PI 4-kinase isozymes account for the major PI kinase activity in many different cell types, and the activity of each isozyme is differentially regulated. The ability to measure and distinguish the activity of individual enzymes is therefore important and forms the subject of the methods in this chapter. We describe the use and application of a versatile radiometric assay to measuring PI 4-kinase activity in a variety of biochemical contexts, from purified enzymes to membrane preparations and permeabilized cells. Until a suitable nonradioactive reagent becomes available, this assay is destined to remain the most widely used method.