rho GDI is an inhibitory GDP/GTP exchange protein for the rho family. Recently, rho GDI has been reported to interact with the GTP-bound form of G25K and rac1 p21 and to inhibit their basal and GTPase-activating protein (GAP)-stimulated GTPase activity. Here, we examined whether rho GDI interacts with the GTP-bound form of rho p21 and rac p21 and inhibits their basal and rho GAP-stimulated GTPase activity, rho GDI interacted with both the GDP- and GTP-bound forms of rhoA p21 and rac1 p21 as estimated by measuring its ability to form a complex with both forms and to inhibit the membrane-binding activity of both forms. The efficiency of rho GDI for interaction with the GTP-bound form was, however, about 10% that for interaction with the GDP-bound form. Moreover, rho GDI inhibited both the basal and rho GAP-stimulated GTPase activity of rhoA p21 and rac1 p21 in a dose-dependent manner. The doses of rho GDI necessary for this action were, however, about 10-fold higher than those necessary for the action to inhibit their GDP/GTP exchange reaction. These results indicate that rho GDI interacts with the GTP-bound form of its substrate small G proteins, as well as with the GDP-bound form, but much less efficiently than with the GDP-bound form.