Synthetic peptides corresponding to the pseudosubstrate domains of protein kinase C (PKC) have been used as specific inhibitors of PKC in in vitro assays and permeabilized cell systems. However, their use in vivo was hampered by the impermeability of the plasma membrane for such peptides. Here, we show that N-myristoylation of the PKC pseudosubstrate nonapeptide Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln permits its use as an inhibitor of PKC in intact cells. The myristoylated peptide, myr-psi PKC, inhibits phosphorylation of the myristoylated alanine-rich C kinase substrate protein, as induced by 12-O-tetradecanoyl-phorbol-13-acetate, and the activation of phospholipase D by bradykinin, which strictly depends on PKC. Half-maximal inhibition is obtained at concentrations of 8 and 20 microM, respectively. An N-myristoylated peptide derived from an inhibitor protein of the cAMP-dependent protein kinases, Myr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp-Ile, was ineffective. These results show that myr-psi PKC is a selective and cell-permeable inhibitor of PKC.