We have analyzed both biochemically and functionally a series of Arf6 mutants, providing new insights into the molecular mode of action of the small G protein Arf6. First, by comparing a fast-cycling mutant (Arf6(T157N)) and a GTPase-deficient mutant (Arf6(Q67L)), we established the necessity for completion of the Arf6 GDP/GTP cycle for recycling of major histocompatibility complex molecules to the plasma membrane. Second, we found that aluminum fluoride (AlF), known for inducing membrane protrusion in cells expressing exogenous wild-type Arf6, stabilized a functional wild-type Arf6.AlF(x) . GTPase-activating protein (GAP) complex in vitro and in vivo. We also found that the tandem mutation Q37E/S38I prevented the binding of two Arf GAPs, but not the effector ARHGAP10, and blocked the formation of membrane protrusion and actin reorganization. Together, our results with AlF(x) and Arf6(Q37E/S38I) demonstrate the critical role of the Arf6 GAPs as effectors for Arf6-regulated actin cytoskeleton remodeling. Finally, competition experiments conducted in vivo suggest the existence of a membrane receptor for GDP-bound Arf6.