The myosin light chain kinase is catalytically inactive unless activated by calmodulin. An autoregulatory pseudosubstrate region located on the carboxyl-terminal side of the enzyme's catalytic domain is responsible for maintaining the enzyme in a latent form. This pseudosubstrate region overlaps the calmodulin binding domain. Synthetic peptides corresponding to the regulatory region can have both substrate antagonist and calmodulin antagonist activities. The pseudosubstrate peptide from the smooth muscle myosin light chain kinase, smMLCK(787-807), S787KDRMKKYMARRKW800QKTGHAV807 is a potent substrate antagonist with a Ki of approximately 12 nM and acts as a calmodulin antagonist with an IC50 = 0.54 microM. The shorter peptide R797RKWQK802, Ki = 1.26 microM, is the core region primarily responsible for substrate antagonist activity and is a weak calmodulin antagonist, IC50 = 181 microM. The corresponding skeletal muscle peptide, KRRWKK was a comparable substrate antagonist, IC50 = 1.63 microM, but a 30-fold more potent calmodulin antagonist, IC50 = 6.1 microM. Substitution of the Trp in either peptide with Phe or Leu did not significantly alter the substrate antagonist activity but markedly reduced calmodulin antagonist activity, RRKWQK, IC50(calmodulin) = 181 microM; RRKFQK, IC50(calmodulin) = 488 microM; RRKLQK, IC50(calmodulin) = 1700 microM; KRRWKK, IC50(calmodulin) = 6.1 microM; KRRLKK, IC50(calmodulin) = 221 microM and KRRFKK, IC50(calmodulin) = 93 microM. The IC50(substrate) values for these peptides ranged from 0.5-13 microM. The peptide KRRLKK was the most selective substrate antagonist and is suitable as an inhibitor for the myosin light chain kinase with the ratio IC50(calmodulin): IC50(substrate) = 273 and an IC50(substrate) = 0.81 microM.