Protein kinase C (PKC) translocation to specific subcellular membrane loci after cellular stimulation is mediated, in part, through its interaction with diacylglycerol, phosphatidylserine, and calcium. Herein, we present multiple lines of evidence which demonstrate that purified rabbit brain PKC undergoes specific acylation with palmitoyl CoA that facilitates its interaction with membrane bilayers. First, incubation of purified rabbit brain PKC with [14C]palmitoyl CoA (5 μM) resulted in the radiolabeling of an 80 kDa band demonstrated by SDS-PAGE and autoradiography, while incubation of PKC with other acyl CoA molecular species (e.g., [3H]myristoyl CoA or [14C]arachidonoyl CoA), fatty acids (e.g., [14C]palmitic and [14C]arachidonic acid), or [14C]diacylglycerol did not result in the incorporation of radiolabel. Second, multiple extractions of [14C]palmitoyl CoA-treated PKC with butanol did not remove the radiolabeled moiety from the 80 kDa PKC band. Third, incubation of the [14C]palmitoyl CoA-radiolabeled PKC moiety with neutral hydroxylamine, hydrochloric acid, or sodium hydroxide released incorporated radiolabel which identified the association between PKC and palmitic acid as a covalent thioester linkage. Fourth, formation of the [14C]palmitoyl CoA-radiolabeled PKC adduct could be prevented by pretreatment of PKC with either dithiobis(nitrobenzoic acid) or N- ethylmaleimide. Fifth, limited trypsinolysis of palmitoylated PKC demonstrated that palmitic acid was exclusively present in the regulatory fragment of PKC without detectable amounts of palmitic acid associated with the catalytic fragment. Sixth, palmitoylated PKC was resolved from its nonpalmitoylated counterpart by Mono Q chromatography, and palmitoylated PKC preferentially associated with cellular membranes while nonpalmitoylated PKC did not. Both palmitoylated and nonpalmitoylated PKC were activated by phosphatidylserine, diacylglycerol, and calcium ion. Collectively, these results demonstrate the acylation of PKC by palmitoyl CoA and identify a novel mechanism which may facilitate the interaction of PKC with biologic membranes.