The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cell. Mg2+ ions play key roles in guanine nucleotide binding and in preserving the structural integrity of GTPases. We describe here the kinetics of the interaction of GTP with the Rho family small GTPase Cdc42 in the absence and presence of Mg2+. In contrast to the cases of Ras and Rab proteins, which require Mg2+ for the nucleotide binding and intrinsic hydrolysis of GTP, our results show that in the absence of Mg-21+the binding affinity of GTP to Cdc42 is in the submicromolar concentration, and the Mg-+(2) ofactor has only a minor effect on the Cdc42-catalyzed intrinsic hydrolysis rate of GTP. These results suggest that the intrinsic GTPase reaction mechanism of Cdc42 may differ significantly from that of other subfamily members of the Ras superfamily. (C) 2002 Elsevier Science (USA). All rights reserved.