The mechanism of the phosphorylation-dependent myosin-linked regulation of smooth muscle and nonmuscle myosins is being investigated. We compared the conventional steady-state kinetic analysis of the actin-activated myosin MgATPase activity with the Nitella-based in vitro motility assay which is a quantitative assay for measuring the velocity of myosin-coated beads over an organized substratum of actin, and analyzed the effect of phosphorylation of various sites on the 20,000 Da light chain of smooth muscle and cytoplasmic myosins. We also determined the effect of tropomyosin on the actin-activated myosin MgATPase activity and on the movement of myosin-coated beads.