Smooth muscle myosin light chain kinase was purified from turkey gizzards. The enzyme was extracted from washed myofibrils and the final step of purification was affinity chromatography using calmodulin coupled to Sepharose 4B. The purified enzyme was characterized with respect to its physical, chemical, and kinetic properties. It has a molecular weight of 130,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 124,000 by sedimentation equilibrium centrifugation under nondenaturing conditions. It is an asymmetric molecule with a Stokes radius of 75 A, a sedimentation coefficient of 4.45 S, and a frictional coefficient of 1.85. Smooth muscle myosin light chain kinase is dependent on the calcium-binding protein calmodulin for activity. It has an apparent K0.5 for calmodulin of 10(-9) M and binds 1 mol of calmodulin/mol of myosin kinase in the presence of calcium. The binding of calmodulin increases the sedimentation coefficient from 4.45 S to 5.05 S, and the Stokes radius from 75 A to 79 A, and does not alter the frictional coefficient. The enzyme has a Km for ATP and the 20,000-dalton light chain of smooth muscle myosin of 50 microM and 5 microM, respectively. It phosphorylates the 20,000-dalton light chain of smooth muscle myosin more rapidly than the equivalent light chain from cardiac and skeletal muscles. It does not phosphorylate histones, alpha-casein, phosphorylase kinase, or phosphorylase b at a significant rate.