A small quantity of unsaturated diacylglycerol (DG) sharply decreased the Ca2+ and phospholipid concentrations needed for full activation of a Ca2+-activated, phospholipid-dependent multifunctional protein kinase described earlier (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T. and Nishizuka, Y. (1979) J. Biol. Chem. 254. 3692-3695). In the presence of unsaturated DG and micromolar order of Ca2+, phosphatidylserine (PS) was most relevant with the capacity to activate the enzyme, whereas phosphatidylethanolamine and phosphatidylinositol (PI) were far less effective. Phosphatidylcholine was practically inactive. It is possible, therefore, that unsaturated DG, which may be derived from PI turnover provoked by various extracellular stimulators, acts as a messenger for activating the enzyme, and that Ca2+ and various phospholipids such as PI and PS seen to play a role cooperatively in this unique receptor mechanism.