A series of synthetic peptides corresponding to the amino-terminal region of chicken gizzard myosin light chain (Mr 20 000) have been tested for their capacity to act as substrates for the cAMP-dependent protein kinase. The 18-residue peptide, K6AKTTK11 K12R13PQRATS19NVFS , was stoichiometrically phosphorylated on serine-19 by the cAMP-dependent protein kinase. This is the same residue phosphorylated by the myosin light chain kinase. The cAMP-dependent protein kinase phosphorylated this peptide with an apparent Km of 120 microM and Vmax of 0.29 mumol . min .-1 mg-1. The Km is 17-fold higher and the Vmax 10-fold lower than the corresponding values obtained with this peptide as substrate for the myosin light chain kinase. The kinetics of phosphorylation of shortened peptides corresponding to this 18-residue sequence together with those of another related sequence, RPQRAKAKTTKATSNVFS , indicated that the myosin light chain kinase had a relatively stronger dependence on lysine residues, whereas the cAMP-dependent protein kinase depends more on arginine residues. Although both the cAMP-dependent protein kinase and the myosin light chain kinase phosphorylate the same serine in the myosin light chain peptides, these enzymes are influenced by different nearby basic residues.