Over time we have come to appreciate that the complex regulation of Rho GTPases involves additional mechanisms beyond the activating role of RhoGEFs, the inactivating function of RhoGAPs, and the sequestering activity of RhoGDIs. For example, Rho GTPases can regulate each other by means of crosstalk signaling and when two Rho proteins activate a common effector. We have recently unraveled a reciprocal mechanism of GTPase regulation wherein one Rho GTPase interacts with two distinct effectors [1]. Spatiotemporal dynamics of Rac1 activity during migration of Dictyostelium cells is apparently regulated by antagonizing interactions of Rac1-GTP with two protein complexes. As shown by monitoring specific fluorescent probes, activated Rac1 is simultaneously present at the leading edge, where it participates in SCAR/WAVE-mediated actin polymerization, and at the trailing edge, where it induces formation of a DGAP1/cortexillin actin- bundling complex. Remarkably, in addition to their opposed localization, the two populations of activated Rac1 also display opposite kinetics of recruitment to the plasma membrane upon stimulation by chemoattractants. We propose that a scaffolding protein, DGAP1, plays roles of an effector and a sequestrator in relation to Rac1, and thereby can provide negative feedback and supersensitivity as prerequisites for spatiotemporal fluctuations in the actin cytoskeleton that are observed in these cells. These findings relating to Rac1 in Dictyostelium suggest a novel principle for regulation of Rho GTPase activity that might also play a role in other cell types and for other Rho family members.