The enzyme, myosin light chain kinase, has been purified to homogeneity from bovine aortic vascular smooth muscle. Approximately 10 mg of enzyme could be obtained from 1 kg of fresh aortas with an overall yield of 26% of the original activity. The vascular myosin light chain kinase has a molecular weight of 160 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Antiserum raised to the aortic myosin light chain kinase in rabbits strongly inhibited phosphotransferase activity. In addition, the antiserum was used to identify myosin kinase in a crude homogenate of vascular smooth muscle by radioimmunoblotting. A single species of the enzyme (Mr = 160 000) was identified. The bovine aortic myosin kinase could be phosphorylated by both cyclic AMP- and GMP-dependent protein kinases. Approximately 2 mols PO4/mole of enzyme could be incorporated by the cyclic AMP-dependent protein kinase in the absence of calmodulin. If Ca2+ and calmodulin were included in the reaction mixture, phosphate incorporation by the cyclic AMP-dependent protein kinase was reduced to 1 mol and phosphorylation by cyclic GMP-dependent protein kinase was completely inhibited. These results were confirmed by tryptic peptide mapping. Two distinct phosphopeptides were identified: site-1 and site-2. Both could be phosphorylated by the cyclic AMP-dependent protein kinase but only site-1 was phosphorylated by the cyclic GMP-dependent enzyme. In the presence of Ca2+ and calmodulin, phosphorylation by cAMP-dependent protein kinase was restricted to site-1. The effect of phosphorylation on myosin light chain kinase activity was determined. Only phosphorylation by cyclic AMP-dependent protein kinase was found to alter the requirement of myosin kinase for calmodulin. The K0.5 (i.e. the concentration of calmodulin required for half-maximal enzyme activation) for calmodulin was 5 nM for the unphosphorylated myosin kinase. With 2 mol PO4/mol myosin kinase incorporated, the K0.5 for calmodulin was increased to 82 nM. When only 1 mol PO4/mol myosin kinase was incorporated, no effect on calmodulin requirement was observed. Moreover, single site phosphorylation had no effect on other activity parameters, including Km for ATP and for light chains. Our studies suggest that cyclic AMP-dependent protein kinase may play an important role in the regulation of vascular myosin kinase activity. Moreover, our results indicate that cyclic GMP-dependent protein kinase does not affect calmodulin-activation of myosin kinase or several other activity parameters.