The acidic phospholipid requirement for protein kinase C (Ca2+/phospholipid-dependent enzyme) activation has been well established, although the molecular nature of this lipid-protein interaction is unclear. The additional requirement for Ca2+ has provided the basis for several models involving charge interactions. We now report that short chain neutral phosphatidylcholines also activate the kinase. Examination of a large series of phosphatidylcholines of varying acyl chain length revealed a close correlation between the ability to form micelles and the ability to support kinase activity. Peak activation occurred in the concentration range just before the critical micelle concentration of each phospholipid. Activation was absolutely dependent on the presence of Ca2+ and diacylglycerol. The possible roles of Ca2+ and phospholipid in the activation process are reexamined in light of these unexpected results.