Recent extensive studies have clarified the functions of the small G protein superfamily, which consists of the Ras, Rho, Rab, Arf, Sar1, and Ran families (for reviews, Refs, 1 and 2). The Ras family regulates gene expression at least through the MAP kinase cascade; the Rho family mainly regulates reorganization of the actin cytoskeleton; the Rab, Arf, and Sar1 families regulate intracellular vesicle trafficking; and the Ran family regulates nuclear transport. Of these cellular functions, reorganization of the actin cytoskeleton, seen in the formation of filopodia, lamellipodia, and ruffles during cell motility, dynamically occurs at specific sites of cells. To regulate this type of dynamic cellular functions, temporal and spatial determination mechanisms of signal transduction would be important. Like other G proteins, small G proteins cycle between the GDP-bound inactive and GTP-bound active forms (1,2). They receive upstream signals through their regulators and transduce signals to downstream targets while they stay in the GTP-bound form. Thus, G proteins serve as timers. There are at least three types of regulators for small G proteins: GDP/GTP exchange protein (GEP) which stimulates conversion from the GDP-bound form to the GTP-bound form; GDP dissociation inhibitor (GDI) which inhibits this reaction; and GTPase activating protein (GAP) which stimulates conversion from the GTP-bound form to the GDP-bound form. Of these regulators, GDI has thus far been found for the Rho and Rab families. We have recently found that the Rho family-Rho GDI system plays an important role in spatial determination in the actin cytoskeletal control (3-6). We briefly describe here this function of the Rho family-Rho GDI system.