We have identified the Rab1B effector-domain mutant (D44N) that, when geranylgeranylated by Rab:geranylgeranyltransferase (GGTase II) in cell-free systems or intact cells, fails to form detectable complexes with GDP-dissociation inhibitors (GDIs). GDI-Rab complexes were collected on anti-FLAG affinity beads after incubating recombinant geranylgeranylated Rab1B with FLAG epitope-tagged GDI in vitro, or transiently coexpressing Myc-tagged Rab1B with FLAG-GDI-alpha or FLAG-GDI-2 in human embryonal kidney 293 cells. [3H]Mevalonate labeling and immunoprecipitation studies confirmed that the inability of Myc-Rab1BD44N to associate with GDI in vivo was not due to failure of the mutant to undergo geranylgeranylation. Immunofluorescence localization and immunoblot analysis of subcellular fractions indicated that expressed Myc-Rab1BD44N was efficiently delivered to intracellular membranes in 293 cells. This was confirmed when the fate of the prenylated pool of Rab1BD44N in 293 cells was traced by labeling the geranylgeranyl groups attached to the nascent protein with [3H]meval onate. However, in contrast to the prenylated Rab1BWT, which was distributed in both the membrane and soluble fractions, the prenylated Rab1BD44N was completely absent from the cytosol. Overexpression of Myc-Rab1BD44N did not impair ER --> Golgi glycoprotein trafficking in 293 cells, which was assessed by monitoring the Golgi-dependent processing of coexpressed beta-amyloid precursor protein. The current findings suggest that nascent prenylated Rab1B can be delivered to intracellular membranes in intact cells without forming a stable complex with GDI, but that recycling of prenylated Rab1B to the cytosolic compartment is absolutely dependent on GDI interaction.