Rab proteins occur in the cytosol bound to Rab-GDP dissociation inhibitor (GDI). We demonstrate here that cytosolic complexes of Rab9 bound to GDI represent a functional pool of Rab9 protein that can be utilized for transport from late endosomes to the trans Golgi network in vitro. Immunodepletion of GDI and Rab proteins bound to GDI led to the loss of cytosol activity; readdition of pure Rab9-GDI complexes fully restored cytosol activity. Delipidated serum albumin could solubilize prenylated Rab9 protein, but unlike Rab9-GDI complexes, Rab9-serum albumin complexes led to indiscriminate membrane association of Rab9 protein. Rab9 delivered to membranes by serum albumin was functional, but GDI increased the efficiency of Rab9 utilization, presumably because it suppressed Rab9 protein mistargeting. Finally, GDI inhibited transport of proteins from late endosomes to the trans Golgi network, likely because of its capacity to inhibit the membrane recruitment of cytosolic Rab9. These experiments show that GDI contributes to the selectivity of Rab9 membrane recruitment and presents functional Rab9 to the endosome-trans Golgi network transport machinery.