The 20,000-dalton light chain of myosin from chicken gizzard has been shown to be phosphorylated in a Ca2+ and calmodulin-independent manner by the activated form of a protease-activated kinase from rabbit reticulocytes. Protease-activated kinase I incorporates phosphate stoichiometrically into the phosphorylatable light chain (P-light chain) in isolated myosin light chains and in actomyosin. The same serine residue appears to be phosphorylated by the protease-activated kinase and the Ca2+-dependent myosin light chain kinase. This conclusion is based on results from two-dimensional peptide maps of chymotryptic and tryptic digests of the phosphorylated P-light chain and from phosphoamino acid analysis of acid hydrolysates. Phosphorylation of the P-light chain by the proteolytically activated protein kinase stimulates the actin-activated Mg-ATPase activity of myosin in the absence of Ca2+. The extent of stimulation of the ATPase activity is similar to that observed upon phosphorylation of actomyosin by the Ca2+-dependent myosin light chain kinase. A proteolytically activated protein kinase with chromatographic properties and substrate specificity similar to protease-activated kinase I from reticulocytes has also been identified in gizzard. Protease-activated kinase I has been shown to be distinct from the Ca2+-dependent myosin light chain kinase by the mode of activation and specificity with other substrates, including phosphorylation of a unique site on myosin P-light chain from skeletal muscle (Tuazon, P. T., Stull, J. T., and Traugh, J. A. (1982) Biochem. Biophys. Res. Commun. 108, 910-917).