Smooth muscle myosin light chain kinase (MLCK) is activated by calcium-calmodulin and, in turn, phosphorylates and activates the smooth muscle actomyosin ATPase, resulting in muscle contraction. The amino acid sequence of the regulatory domain of MLCK is known, and it contains a region that binds calmodulin and also bears a strong homology to the phosphorylation site in the substrate. Thus, it has been called the "pseudosubstrate". It has been proposed that calmodulin activates MLCK by binding to and reversing the autoinhibitory function of the pseudosubstrate. Synthetic peptides based on this sequence inhibit MLCK both by binding to calmodulin and by competing with the substrate at the active site. In the work reported here, we have synthesized a large number of peptides from the regulatory region of MLCK (MLCK 480-516). The region was systematically analyzed by dividing it into fragments of two to six amino acids, each containing one or more basic residues, in order to map in detail the calmodulin binding site and the autoinhibitory region. It was observed that both calmodulin binding and autoinhibition are mediated by several different fragments of the regulatory sequence. Two nonoverlapping peptides, MLCK 480-493 and MLCK 494-504, are similar in potency in inhibiting the enzyme (IC50's of 2 and 6 microM, respectively). Larger fragments, combining multiple inhibitory regions, are more potent inhibitors. For example, MLCK 480-504 is extremely potent, with an IC50 of 13 nM. The calmodulin binding site and active site directed inhibitory regions overlap, but are not identical. Residues 505-512 are important only for calmodulin binding.