The mammalian counterpart of the yeast ras p21 GDP/GTP exchange protein CDC25, ras GRF, was expressed in Escherichia coli and purified, and its kinetic properties were compared with those of another mammalian ras p21 GDP/GTP exchange protein, smg GDS. ras GRF was active on Ki- and Ha-ras p21s but inactive on rap1A p21, rhoA p21, rac1 p21, and rab3A p25, whereas smg GDS was active on Ki-ras p21, rap1A p21, rhoA p21, and rac1 p21 but inactive on Ha-ras p21 and rab3A p25. The Kcat values of ras GRF and smg GDS for Ki-ras p21 as a common substrate were calculated to be 1.2 and 0.37 nmol/min/nmol, respectively. The Km values of ras GRF and smg GDS for Ki-ras p21 were 680 and 220 nM, respectively. rasGRF was slightly active on post-translationally unprocessed Ki-ras p21 but much more effective on post-translationally processed Ki-ras p21 than on post-translationally unprocessed Ki-ras p21. smg GDS was active on post-translationally processed Ki-ras p21 but inactive on post-translationally unprocessed Ki-ras p21. Moreover, as described for smg GDS, ras GRF showed a potency to inhibit the binding of Ki-ras p21 to membrane and to induce the dissociation of prebound Ki-ras p21 from the membrane. These results indicate that ras GRF and smg GDS show apparently similar kinetic properties except for the different substrate specificities and the requirement of the post-translational processing of Ki-ras p21.