Myosin light-chain kinase (MLCK) comprised of N-terminal actin-binding domain, central catalytic domain, and C-terminal myosin-binding domain. It exerted not only kinase activity to phosphorylate 20 kDa regulatory light chain of smooth muscle but also exerted non-kinase activity on myosin motor and myosin ATPase activities (Nakamura et al., Biochem. Biophys. Res. Commun. 2008, 369, 135). The previous studies on the multiple MLCK functions were done using MLCK fragments. The present study reported the expression of whole MLCK molecules in Escherichia coli in a large amount. The construct in which the calmodulin (CaM) binding domain for regulating kinase activity was mutated lost the kinase activity. However, the mutant exerted non-kinase activity and inhibited both myosin motor and ATPase activities. The domain that regulated kinase activity was also shown to be involved in the Ca(2+) regulation of non-kinase activity. The deletion mutants of actin-binding domain which located at N-terminal 1-41 amino acids demonstrated that non-kinase activity was mediated through actin filaments.