A crude myosin fraction from bovine brain has been found to contain a Ca2+-independent myosin kinase that catalyzes the phosphorylation of 20,000-Da light chain of gizzard myosin. The myosin kinase has been separated from the myosin by Sepharose CL-4B gel filtration and purified further by chromatography on phosphocellulose, Sephacryl S-300, and hydroxylapatite. The myosin kinase was found to copurify with casein kinase II and show the same substrate specificity with the casein kinase. These results indicate that the myosin kinase is identical to casein kinase II. The purified myosin kinase catalyzed the preferential phosphorylation of the threonyl residues of 20,000-Da light chains of gizzard and brain myosins. The 17,000-Da light chains of these myosins and the mixed light chains of skeletal and cardiac muscle myosins were not phosphorylated by the enzyme to an appreciable extent.