Abstract: Monitoring Editor: Daniel Lew The small GTP-binding proteins of the Rho family are implicated in various cell functions including establishment and maintenance of cell polarity. Activity of Rho GTPases is not only regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) but also by guanine nucleotide dissociation inhibitors (GDIs). These proteins have the ability to extract Rho proteins from membranes and keep them in an inactive cytosolic complex. Here, we show that Rdi1, the sole Rho GDI of the yeast S. cerevisiae, contributes to pseudohyphal growth and mitotic exit. Rdi1 interacts only with Cdc42, Rho1 and Rho4, and regulates these Rho GTPases by distinct mechanisms. Binding between Rdi1 and Cdc42 as well as Rho1 is modulated by the Cdc42 effector and p21-activated kinase (PAK) Cla4. Following membrane extraction mediated by Rdi1, Rho4 is degraded by a novel mechanism, which includes the glycogen synthase kinase 3beta (GSK-3beta) homolog Ygk3, vacuolar proteases and the proteasome. Taken together, these results indicate that Rdi1 uses distinct modes of regulation for different Rho GTPases.