Myosin light chain kinase (MLCK) was prepared from smooth muscle of bovine aorta. MLCK inhibited the ATP-dependent movement of actin filaments on a glass surface coated with smooth muscle myosin that had been phosphorylated. The inhibitory effect was abolished by calmodulin in the presence of Ca2+ (Ca-CaM). The abolition was also observed when the concentration of actin filaments was increased. The inhibitory effect and its abolition were related to the actin-binding activity of MLCK, that is antagonized by Ca-CaM.