Ras-catalyzed guanosine 5′ triphosphate (GTP) hydrolysis proceeds through a loose transition state as suggested in our previous study of 18O kinetic isotope effects (KIE) [Du, X. et al. ((2004) ) Proc. Natl. Acad. Sci. U.S.A. 101, 8858-8863]. To probe the mechanisms of GTPase activation protein (GAP)-facilitated GTP hydrolysis reactions, we measured the 18O KIEs in GTP hydrolysis catalyzed by Ras in the presence of GAP334 or NF1333, the catalytic fragment of p120GAP or NF1. The KIEs in the leaving group oxygens (the β nonbridge and the β-γ bridge oxygens) reveal that chemistry is rate-limiting in GAP334-facilitated GTP hydrolysis but only partially rate-limiting in the NF1333-facilitated GTP hydrolysis reaction. The KIEs in the γ nonbridge oxygens and the leaving group oxygens reveal that the GAP 334 or NF1333-facilitated GTP hydrolysis reaction proceeds through a loose transition state that is similar in nature to the transition state of the GTP hydrolysis catalyzed by Ras alone. However, the KIEs in the pro-S β, pro-R β, and β-γ oxygens suggest that charge increase on the β-γ bridge oxygen is more prominent in the transition states of GAP334-and NF1333-facilitated reactions than that catalyzed by the intrinsic GTPase activity of Ras. The charge distribution on the two β nonbridge oxygens is also very asymmetric. The catalytic roles of active site residues were inferred from the effect of mutations on the reaction rate and KIEs. Our results suggest that the arginine finger of GAP and amide protons in the P-loop of Ras stabilize the negative charge on the β-γ bridge oxygen and the pro-S β nonbridge oxygen of a loose transition state, whereas Lys-16 of Ras and Mg2+ are only involved in substrate binding.