The Rho subfamily, consisting of three members (RhoA, -B and -C), belongs to the small GTP-binding protein superfamily. The Rho subfamily is implicated in regulation of various actin filament-dependent cell functions, such as cell aggregation, cell motility and cytokinesis. The Rho subfamily receives an upstream signal and is converted from the GDP-bound inactive form to the GTP-bound active form which transduces a signal to a downstream pathway. This conversion is regulated by GDP/GTP exchange proteins (GEPs) and several GEPs for the Rho subfamily have been identified. The GEPs thus far reported are mainly isolated from the cytosol fraction of various tissues and are not specific for the Rho subfamily. Here we have partially purified a membrane-associated GEP specific for the Rho subfamily (mRho GEP). mRho GEP was extracted from the crude synaptic membrane fraction of rat brain by a combination of detergent and NaCl, and partially purified by several column chromatographies. The partially purified mRho GEP was active on RhoA but was inactive on other small GTP-binding proteins including at least Rac1, Ki-Ras and Rab3A. RhoA undergoes post-translational lipid modifications and mRho GEP required these lipid modifications for its GEP activity. mRho GEP was not active in the presence of Rho GDI, an inhibitory Rho GEP. These results indicate that there is a membrane-associated GEP specific for Rho and suggest that Rho is activated by this GEP on the membranes.