The roles of specific and nonspecific interactions in the regulation of protein kinase C by lipid have been examined. Binding and activity measurements reveal two mechanisms by which protein kinase C interacts with membranes: (1) a specific binding to the activating lipid phosphatidylserine and (2) a nonspecific binding to nonactivating, acidic lipids. The specific interaction with phosphatidylserine is relatively insensitive to ionic strength, surface charge, and the presence of nonactivating lipids. The two second messengers of the kinase, diacylglycerol and Ca2+, increase markedly the affinity of the kinase for phosphatidylserine. In contrast, the nonspecific interaction is sensitive to ionic strength and surface charge, and is unaffected by diacylglycerol. These results suggest that electrostatic interactions promote the binding of protein kinase C to membranes but the cooperative and selective binding of phosphatidylserine is the dominant driving force in a productive protein-lipid interaction.