The syntheses of a protein kinase C (PKC) peptide substrate, H‐Lys‐Arg‐Thr‐Leu‐Arg‐OH, and a phosphopeptide analog of the synthetic substrate, H‐Lys‐Arg‐Thr(P)‐Leu‐Arg‐OH, are reported. PKC phosphorylates the peptide with an apparent KMof 0.30 ± 0.04 mM and an apparent Vmaxequal to one‐tenth that of histone III‐S. The synthesis of the phosphopeptide features a recently developed convenient phosphorylation procedure for serine and threonine usingN,N‐diethylamino‐dibenzylphosphoramidite. A complete characterization of the PKC substrate and its corresponding phosphopeptide b