Guanine nucleotide dissociation inhibitor (GDI) regulates the recycling of Rab GTPases involved in vesicle targeting and fusion. We have analyzed the requirement for conserved amino acid residues in the binding of Rab1A and the function of GDI in transport of cargo between the endoplasmic reticulum (ER) and the Golgi apparatus. Using a new approach to monitor GDI-Rab interactions based on the change in fluorescence associated with the release of methylanthraniloyl guanosine di(tri)phosphate-GDP (mGDP) from Rab, we show that residues previously implicated in the binding of the synapse-specific Rab3A, including Gln-236, Arg-240, and Thr-248, are essential for the binding of Rab1A. Mutation of each of these residues has potent effects on the ability of GDI to remove Rab1A from membranes and inhibit ER to Golgi transport in vitro. Given the sequence divergence between Rab1A and 3A (35% identity), these residues are proposed to play a general role in GDI function in the cell. In contrast, several other residues found within or flanking the Rab-binding region were found to have differential effects in the recognition and recycling of Rab1A and 3A, and therefore direct selective interaction of GDI with individual Rab proteins. Intriguingly, mutation of one residue, Arg-70, led to a reduction of Rab1A binding, failed to extract Rab1A from membranes in vitro, yet bound membranes tightly and potently inhibited ER to Golgi transport. These results provide evidence that novel membrane-associated factor(s) mediate Rab-independent GDI interaction with membranes.