The study investigates the role of Rho-associated serine/threonine kinase (Rho-kinase) in the phosphorylation and activation of myosin light chain (MLC). Rho-kinase, activated by GTP-Rho, was found to stoichiometrically phosphorylate MLC at Ser-19, the same site phosphorylated by MLC kinase. The phosphorylation of MLC by Rho-kinase increased the actin-activated MgATPase activity of myosin, suggesting that Rho-kinase plays a crucial role in regulating muscle contraction and other physiological functions associated with actin-myosin interactions. The findings indicate that Rho-kinase may contribute to the regulation of cytokinesis, cell motility, and smooth muscle contraction by phosphorylating MLC and activating myosin.The study investigates the role of Rho-associated serine/threonine kinase (Rho-kinase) in the phosphorylation and activation of myosin light chain (MLC). Rho-kinase, activated by GTP-Rho, was found to stoichiometrically phosphorylate MLC at Ser-19, the same site phosphorylated by MLC kinase. The phosphorylation of MLC by Rho-kinase increased the actin-activated MgATPase activity of myosin, suggesting that Rho-kinase plays a crucial role in regulating muscle contraction and other physiological functions associated with actin-myosin interactions. The findings indicate that Rho-kinase may contribute to the regulation of cytokinesis, cell motility, and smooth muscle contraction by phosphorylating MLC and activating myosin.