Myosin light chain kinase (MLCK) is a multifunctional regulatory protein of smooth muscle contraction, which includes an N-terminal actin-binding domain, central catalytic domain, calmodulin-binding domain, and a C- terminal myosin-binding domain. Myosin phosphorylated by the catalytic domain of MLCK is in an active form and interacts with the actin filament to contract smooth muscle. This mode of phosphorylation is widely accepted as the regulatory mechanism for actin-myosin interaction. However, there are a number of observations that are not explained by this mechanism. An MLCK C-terminal fragment (MLCK fragment) containing the myosin-binding domain have been previously engineered but devoid of a catalytic domain, which has confirmed how myosin is stimulated by this non-kinase pathway. A recombinant GST-fusion protein of the MLCK fragment was expressed in E.coli and detected by SDS-PAGE. Through Glutathione-Sepharose 4B affinity chromatography, a single pure band of the MLCK fragment was obtained. The phosphorylated myosin ATPase activities of the MLCK fragment, as well as its proteolytic fragment HMM and S1 were measured with the EnzChek Phosphate Assay Kit. The MLCK fragment stimulated phosphorylated myosin ATPase activity (V_max=(19.426±1.669)-fold, K_m=(0.486±0.106)μmol/L). Similar stimulation figures were obtained by measuring the ATPase activity of phosphorylated HMM and S1. The data suggests that MLCK could activate phosphorylated myosin, HMM and S1. In addition, in vitro motility assays demonstrated that increasing amounts of the MLCK fragment increased actin-myosin interaction and sliding. Also, the velocity of actin filaments could be enhanced on a gizzard smooth muscle myosin surface with the MLCK fragment. It is conclude that the non-kinase C-terminal domain of MLCK is independent of the phosphorylating mode for activation of myosin.