Previous work has established that the integrin signal transduction pathway plays an important role in regulation of epithelial tubule formation. Furthermore, it has been demonstrated that Rho-kinase, an effector of the Rho signaling pathway is an important downstream modulator of integrin-regulated renal and mammary epithelial tubule morphogenesis. In studies presented here, MDCK cells that expressed mutant Rho family GTPases (dominant-negative, constitutively active) were used to provide further insight into Rho GTPase signaling and regulation of epithelial tubule formation. Using collagen gel overlays on MDCK cells as a model system, phosphorylated myosin light chain (MLC) was observed at the leading edge of migrating lamellipodia. This epithelial remodeling led to formation of multicellular branching epithelial tubules with extensive tight junctions. However, in cells expressing dominant-negative RhoN19, MLC phosphorylation, epithelial remodeling and epithelial tubule formation were inhibited. Instead, only small apical lumens with a solitary tight junctional ring were observed providing further evidence that Rho signaling through Rho-kinase was important in regulation of epithelial tubule formation. Since the present model for the Rho signaling pathway proposes that Rac plays a prominent but reciprocal role in cell regulation, experiments were done using cells that expressed constitutively active RacV12. When incubated with collagen gels, RacV12 expressing cells formed small apical lumens with disrupted tight junctions. Complementary experiments with wild type MDCK cells demonstrated that endogenous Rac1 activation levels decreased over a time course consistent with lamellipodia and tubule formation. Under these conditions, Rac1 was initially localized to the basolateral membrane. However, after epithelial remodeling Rac1 was observed primarily in lamellipodia. These studies support a model in which Rac1 and RhoA are important modulators of cell motility and junctional complex integrity during epithelial tubule formation.