Ras family GTPases play central roles in a wide variety of biological responses, including cell proliferation, differentiation, and oncogenic transformation. We searched for novel guanine nucleotide exchange factors of HRas and isolated small G-protein dissociation stimulator (smgGDS), a guanine nucleotide exchange factor known to act on numerous Ras and Rho family GTPases. SmgGDS specifically interacts with both dominant negative and nucleotide free forms of H and NRas, but not with the corresponding oncogenic forms. An effector domain mutant of HRas, HRasN17G37, selectively lost the ability to bind smgGDS. However, smgGDS does not catalyze guanine nucleotide exchange on either H or NRas in vitro. In contrast, substrates of smgGDS, such as KRas, Rac1, and RhoA, bind to smgGDS in both active and inactive forms which requires the presence of poly-basic residues in the C-termini of the GTPases. Our data suggest that the C-terminal poly-basic region of small GTPases is important for both binding and nucleotide exchange by smgGDS. Furthermore, these data underscore the idea that mammalian Ras isoforms are not functionally equivalent.