Mechanisms of guanosine triphosphate (GTP) hydrolysis by members of the G protein α subunit-p21ras superfamily of guanosine triphosphatases have been studied extensively but have not been well understood. High-resolution x-ray structures of the GTP-γS and GDP·AlF 4- complexes formed by the G protein Giα1 demonstrate specific roles in transition-state stabilization for two highly conserved residues. Glutamine204 (Gln61 in p21 ras) stabilizes and orients the hydrolytic water in the trigonal-bipyramidal transition state. Arginine 178 stabilizes the negative charge at the equatorial oxygen atoms of the pentacoordinate phosphate intermediate. Conserved only in the Gα family, this residue may account for the higher hydrolytic rate of Gα proteins relative to those of the p21ras family members. The fold of G iα1 differs from that of the homologous Gtα subunit in the conformation of a helix-loop sequence located in the α-helical domain that is characteristic of these proteins; this site may participate in effector binding. The amino-terminal 33 residues are disordered in GTPγS-Giα1, suggesting a mechanism that may promote release of the βγ subunit complex when the a subunit is activated by GTP.