THE Rab GTPases are key regulators of vesicular transport(1-6). A fraction of Rab proteins is present in the cytosol, bound with GDP, complexed tb a protein termed GDI(7-10). Rab9 is localized primarily to late endosomes, where it aids the transport of mannose 6-phosphate receptors to the trans-Golgi network(11). It has been proposed that Rab proteins are delivered to specific membranes by GDI, and that this process is accompanied by the exchange of bound GDP for GTP(1-3). In addition, Rab localization requires carboxy-terminal prenylation and specific structural determinants(12-14). Here we describe the reconstitution of the selective targeting of prenylated Rab9 protein onto late endosome membranes and show that this process is accompanied by endosome-triggered nucleotide exchange.