Previous reports using various protein kinase inhibitors have suggested that protein kinase activity is necessary for both the induction and maintenance of hippocampal long-term potentiation (LTP), a cellular phenomenon likely to contribute to mammalian memory formation. We designed and characterized a selective peptide substrate for protein kinase C (PKC), corresponding to amino acids 28 to 43 of the neuronal protein neurogranin, and used the substrate to obtain direct biochemical evidence for activation of PKC in both the induction and maintenance phases of LTP. As the effect cannot be accounted for by either of two well-known mechanisms for persistent PKC activation, membrane insertion, or proteolysis, the persistent activation of PKC in the maintenance phase of LTP appears to occur via another mechanism. The maintenance phase of LTP is associated with decreased immunoreactivity of PKC, an effect that can be reversed with phosphatase treatment. Thus, PKC appears to be both phosphorylated and persistently activated in the maintenance phase of LTP.