Post-translationally isoprenylated proteins represent major hubs in most membrane-connected signaling networks. GDP dissociation inhibitors (GDIs) are molecular chaperones that shuttle geranylgeranylated GTPases between membranes and the cytosol. Despite numerous studies, the mechanism of targeted membrane delivery of GTPases remains unknown. Here we have combined chemical synthesis and expressed protein ligation to generate fluorescent lipidated RabGTPase-based sensor molecules. Using these protein probes, we have demonstrated that RabGDI and the related Rab escort protein REP show a three-order-of-magnitude greater affinity for GDP-bound Rab GTPase than for the GTP-bound state. Combined with a relatively high dissociation rate of the Rab–GDI complex, this would enable guanine nucleotide exchange factors (GEFs) to efficiently dissociate the complex and promote membrane attachment of the GTPase. The findings suggest strongly that GEFs are necessary and sufficient for membrane targeting of GTPases and that the previously proposed GDI displacement factors (GDFs) are not thermodynamically required for this process.