The substrate specificity of the multifunctional calmodulin-dependent protein kinase from skeletal muscle has been studied using a series of synthetic peptide analogs. The enzyme phosphorylated a synthetic peptide corresponding to the NH2-terminal 10 residues of glycogen synthase, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-NH2, stoichiometrically at Ser-7, the same residue phosphorylated in the parent protein. The synthetic peptide was phosphorylated with a Vmax of 12.5 mumol X min-1 X mg-1 and an apparent Km of 7.5 microM compared to values of 1.2 mumol X min-1 X mg-1 and 3.1 microM, respectively, for glycogen synthase. Similarly, a synthetic peptide corresponding to the NH2-terminal 23 residues of smooth muscle myosin light chain was readily phosphorylated on Ser-19 with a Km of 4 microM and a Vmax of 5.4 mumol X min-1 X mg-1. The importance of the arginine 3 residues NH2-terminal to the phosphorylated serine in each of these peptides was evident from experiments in which this arginine was substituted by either leucine or alanine, as well as from experiments in which its position in the myosin light chain sequence was varied. Positioning arginine 16 at residues 14 or 17 abolished phosphorylation, while location at residue 15 not only decreased Vmax 14-fold but switched the major site of phosphorylation from Ser-19 to Thr-18. It is concluded that the sequence Arg-X-Y-Ser(Thr) represents the minimum specificity determinant for the multifunctional calmodulin-dependent protein kinases. Studies with various synthetic peptide substrates and their analogs revealed that the specificity determinants of the multifunctional calmodulin-dependent protein kinase were distinct from several other "arginine-requiring" protein kinases.