Myosin II is a critical regulator of cell migration that generates polarity, controls protrusion, and promotes adhesion maturation and retraction of the rear Myosin II has an ATPase motor domain that is regulated by phosphorylation of the regulatory light chain (RLC) on Thr18 and Ser19 Here, we address the activation and specific function of the two phosphorylation states of the RLC, mono- (S19) and/or diphosphorylation (T18 + 519), in cell polarity and adhesion Specific phospho-antibodies reveal that adhesion to fibronectin via the alpha 5 beta 1 integrin promotes localized mono- and di-phosphorylation of the RLC that follow different kinetics Using phospho-mimetic mutants, we show that mono-phosphorylation promotes adhesion maturation in protrusions resulting in focal adhesions throughout the cell In contrast, di-phosphorylation produces large, stable actomyosin bundles and large, non-dynamic adhesions that define the rear Finally, RLC phosphorylation regulates the assembly and stability of MIIB. but not MIIA Our data reveal a novel mechanism by which the degree of phosphorylation of the RLC differentially controls cell adhesion and polarity (C) 2010 Elsevier Inc All rights reserved