The mechanisms that control organization of endothelial cells (ECs) into new blood vessels are poorly understood. We hypothesized that the GTPase Rho, which regulates cytoskeletal architecture, is important for EC organization during neovascularization. To test this hypothesis, we designed a highly versatile mouse skin model that used vascular endothelial growth factor-expressing cells together with packaging cells producing retroviruses encoding RhoA GTPase mutants. In this animal model, dominant negative N19RhoA selectively impaired assembly of ECs into new blood vessels; and, in contrast, active V14RhoA stimulated ECs to form blood vessels with functional lumens. In vitro, dominant negative N19RhoA reduced EC actin stress fibers and prevented ECs from contracting and reorganizing into precapillary cords within collagen gels. In contrast, active V14RhoA promoted EC stress fiber formation, contractility, and organization into cords. Neither N19RhoA nor V14RhoA significantly affected EC proliferation or migration in vitro; and, similarly, neither mutant significantly affected EC density during angiogenesis in vivo. Thus, these studies identify a critical and selective role for Rho activity in regulating EC assembly into new blood vessels, and they identify both negative and positive manipulation of Rho activity, respectively, as strategies for suppressing or promoting the organizational stages of neovascularization.