Objective-: RhoJ/TCL was identified by our group as an endothelial- expressed Rho GTPase. The aim of this study was to determine its tissue distribution, subcellular localization, and function in endothelial migration and tube formation. Methods and Results-: Using in situ hybridization, RhoJ was localized to endothelial cells in a set of normal and cancerous tissues and in the vasculature of mouse embryos; endogenous RhoJ was localized to focal adhesions by immunofluorescence. The proangiogenic factor vascular endothelial growth factor activated RhoJ in endothelial cells. Using either small interfering (si)RNA-mediated knockdown of RhoJ expression or overexpression of constitutively active RhoJ (daRhoJ), RhoJ was found to positively regulate endothelial motility and tubule formation. Downregulating RhoJ expression increased focal adhesions and stress fibers in migrating cells, whereas daRhoJ overexpression resulted in the converse. RhoJ downregulation resulted in increased contraction of a collagen gel and increased phospho-myosin light chain, indicative of increased actomyosin contractility. Pharmacological inhibition of Rho-kinase (which phosphorylates myosin light chain) or nonmuscle myosin II reversed the defective tube formation and migration of RhoJ knockdown cells. Conclusion-: RhoJ is endothelial-expressed in vivo, activated by vascular endothelial growth factor, localizes to focal adhesions, regulates endothelial cell migration and tube formation, and modulates actomyosin contractility and focal adhesion numbers. Copyright © 2011 American Heart Association. All rights reserved.