Myosin light chain kinase was prepared from rabbit skeletal muscle. DEAE-Sephadex, calmodulin-Sepharose 4B affinity gel and Ultrogel AcA 34 were used for the purification. It took 3 days for the preparation, and 6.2 mg of myosin light chain kinase was isolated from 600 g of frozen muscle. The molecular weight of the myosin light chain kinase estimated by sedimentation equilibrium analysis was 103,000 +/- 4,100. The isoelectric point was 5.0. Chemical modification of cysteine residues did not affect the catalytic activity, but modification of tyrosine residues diminished the activity. In order to activate myosin light chain kinase, it was necessary to bind calmodulin in an equimolar ratio and the dissociation constant was estimated to be 3.6 nM. The optimum pH for the catalytic activity was 7.5, and the activity was inhibited by NaCl and KCl. In the presence of 2.74 mg/ml myosin light chain and 75 mM KCl, the catalytic activity was found to be 88 s-1. The Vm and Km at 0.14 M KCl were 100 s-1 and 53 microM, respectively, for the isolated light chain as substrate and 70-80 s-1 and 19 microM for myosin as substrate.