Diacylglycerol-induced translocation of diacylglycerol kinase (ATP:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107) from the soluble to the membrane-bound compartments was demonstrated both in crude tissue homogenates and in a reconstituted enzyme-membrane model system. In homogenates of either rat brain or liver, incubation with diacylglycerol or phospholipase C, but not phospholipase A2 or phospholipase D, resulted in the translocation of diacylglycerol kinase activity from the soluble to the particulate fraction. This observation formed the basis for the first step in a two-step purification of diacylglycerol kinase. Enzyme extracted in 1 M salt from membranes of rat brain homogenates made in the presence of phospholipase C was purified further by affinity chromatography on a column containing phosphatidylserine, diacylglycerol, and cholesterol immobilized in polyacrylamide. This step yielded an enzyme preparation (step 2 enzyme) that was 500-to 750-fold purified (relative to the tissue homogenate) and required phosphatidylserine for stability. All other lipids tested failed to stabilize the enzyme. The properties of the enzyme preparation were similar to those of mammalian diacylglycerol kinases described by others. Reconstitution experiments showed that the soluble step 2 enzyme bound to inside-out vesicles of human erythrocytes only in the presence of diacylglycerol or phospholipase C but not phospholipase A2 or D. Redistribution of the kinase from soluble to vesicle-bound forms occurred rapidly and was dependent on the concentration of phospholipase C used to treat the vesicles. Physiological concentrations of calcium (50-1000 nM) did not enhance the phospholipase C-mediated translocation of the kinase. Thus, diacylglycerol kinase can translocate from cytosol to membranes in a manner dependent on the content of membrane-bound diacylglycerol but independent of the ambient concentration of calcium.The recent advent of diacylglycerol as the endogenous activator ofprotein kinase C has propelled diacylglycerol into the limelight as an important second messenger, coupling receptor-mediated hydrolysis of phosphatidylinositols to activation of protein kinase C. It has been known for some time that diacylglycerol is produced as a result of the hydrolysis of phosphatidylinositols by phospholipase C (1-3). Many reports (4-8) indicate that protein kinase C can translocate from cytosol to membranes as a result of the formation of diacylglycerol and the increased cytosolic calcium concentration resulting from inositol trisphosphate formation. However, diacylglycerol is rapidly converted to phosphatidate by diacylglycerol kinase (ATP:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107) (9, 10). Like protein kinase C, diacylglycerol kinase requires phospholipid for catalytic function (11, 12). Moreover, because diacylglycerol kinase is at least partially responsible for the half-life of diacylglycerol, it may play an important role in regulating the coupling of agonist-induced generation of membrane diacy...