Using the specific antibodies pLC1 and pLC2 for mono- and diphosphorylated 20-kDa myosin light chain (MLC20) at Ser19and at both Thr18and Ser19, respectively, we visualized the dynamics of the MLC20phosphorylation in rabbit aortic smooth muscle cells (cell line SM-3) stimulated with PGF2α. In the resting state, the diphosphorylated form was located in the peripheral region of the cell, such as the leading edge or the adhesion plaque, and the monophosphorylated form was located not only in the peripheral region but also on a discontinuous fibrillary structure along the long axis of the cell. After stimulation with 30 μM PGF2α, although localization of the monophosphorylated form changed little, the content of the diphosphorylated form increased and the distribution spread along the fibrillary structure to an extent the same as or similar to that of the monophosphorylated form, which colocalized with actin filament bundles. The diphosphorylation of MLC20was more sensitive to protein kinase inhibitors, HA-1077, HA-1100, staurosporine, wortmannin, and ML-9, than was the monophosphorylation. In light of these observations, we propose that MLC20diphosphorylation and monophosphorylation are regulated by different mechanisms.