Rho family small GTPases are critical regulators of multiple cellular functions. Dbl-homology-domain-containing proteins are the classical GEFs (guanine nucleotide exchange factors) responsible for activation of Rho proteins. Zizimin1 is a Cdc42-specific GEF that belongs to a second family of mammalian Rho-GEFs, CZH [CDM (Ced-5/DOCK180/Myoblast city)-zizimin homology] proteins, which possess a novel type of GEF domain. CZH proteins can be divided into a subfamily related to DOCK 180 and a subfamily related to zizimin1. The two groups share two conserved regions named the CZH1 (or DHR1) domain and the CZH2 (DHR2 or DOCKER) domains, the latter exhibiting GEF activity. We now show that limited proteolysis of zizimin1 suggests the existence of structural domains that do not correspond to those identified on the basis of homologies. We demonstrate that the N-terminal half binds to the GEF domain through three distinct areas, including the CZH1, to inhibit the interaction with Cdc42. The N-terminal PH (pleckstrin homology) domain binds phosphoinositides and mediates zizimin1 membrane targeting. These results define two novel functions for the N-terminal region of zizimin1.