A variety of contractile stimuli increase actin polymerization, which is essential for smooth muscle contraction. However, the mechanism(s) of actin polymerization associated with smooth muscle contraction is not fully understood. We tested the hypothesis that phosphorylated myosin triggers actin polymerization. The present study was conducted in isolated intact or -escin permeabilized rat small mesenteric arteries. Reductions in the 20 kD myosin regulatory light chain (MLC₂₀) phosphorylation were achieved by inhibiting myosin light chain kinase (MLCK) with ML-7. Increases in MLC₂₀ phosphorylation were achieved by inhibiting myosin light chain phosphatase (MLCP) with microcystin. Isometric force, the degree of actin polymerization as indicated by F/G-actin ratio, and MLC₂₀ phosphorylation were determined. Reductions in MLC₂₀ phosphorylation were associated with decreased force development and actin polymerization. Increased MLC₂₀ phosphorylation was associated with increased force generation and actin polymerization. We also found that a heptapeptide that mimics the actin-binding motif of myosin II enhanced microcystin-induced force generation and actin polymerization without affecting MLC₂₀ phosphorylation in -escin permeabilized vessels. Collectively our data demonstrates that MLC₂₀ phosphorylation is capable of triggering actin polymerization. We further suggest that the binding of myosin to actin triggers actin polymerization and enhances force development in arterial smooth muscle.