Phosphorylation of the myosin regulatory light chains (RLCs) activates contraction in smooth muscle and modulates force production in striated muscle. RLC phosphorylation changes the net charge in a critical region of the N terminus and thereby may alter interactions between the RLC and myosin heavy chain. A series of N-terminal charge mutations in the human smooth muscle RLC has been engineered, and the mutants have been evaluated for their ability to mimic the phosphorylated form of the RLC when reconstituted into scallop striated muscle bundles or into isolated smooth muscle myosin. Changing the net charge in the region from Arg-13 to Ser-19 potentiates force in scallop striated muscle and maintains smooth muscle myosin in an unfolded filamentous state without affecting ATPase activity or motility of smooth muscle myosin. Thus, the effect of RLC phosphorylation in striated muscle and its ability to regulate the folded-to-extended conformational transition in smooth muscle may be due to a simple reduction of net charge at the N terminus of the light chain. The ability of phosphorylation to regulate smooth muscle myosin's ATPase activity and motility involves a more complex mechanism.