This work studies the coordination of the action of GTPase-activating protein (GAP) and guanine nucleotide exchange factor (GEF) on activated human c-Ha-Ras p21. Purified human p120-GAP was obtained with a new efficient procedure. To distinguish the GTPase-activating effect of p120-GAP from other effects dependent on the interaction with activated Ha-Ras, the nonhydrolyzable GTP analogue guanosine 5'-O-(thiotriphosphate) (GTPgammaS) was used. The results showed that the GTPgammaS/GTPgammaS exchange enhanced by the C-terminal catalytic domain of the yeast GEF Sdc25p (C-Sdc25p) is prevented by p120-GAP. This effect is strictly specific for the activated form of Ha-Ras, the target of GAP; no effect on Ha-Ras.GDP was detectable. The GAP catalytic domain also inhibited C-Sdc25p but to a lower extent. The interfering effect by p120-GAP was also evident in a homologous mammalian system, using full-length mouse RasGEF, its C-terminal half-molecule, or C-terminal catalytic domain. As a consequence of this inhibition, presence of p120-GAP enhanced the regeneration of Ha-Ras.GTPgammaS by GEF at a GDP:GTPgammaS ratio mimicking the in vivo GDP:GTP ratio. Our work describes a novel function of p120-GAP and suggests a mechanism by which GAP protects Ha-Ras.GTP in vivo against unproductive exchanges. This constrain is likely involved in the regulation of the physiological GDP/GTP cycle of Ras and in the action of p120-GAP as downstream effector of Ras. Helix alpha3 is proposed as a Ras element playing a key-role in the interference between GAP and GEF on Ras.