GDIs (GDP-dissociation inhibitors) bind to Rab GTPases and mediate their membrane targeting and recycling. In vitro, most Rabs can bind to either of the major isoforms of GDI, leading to the assumption that the proportion of each specific Rab/GDI complex in vivo reflects the relative abundance of the alpha versus beta forms of GDI. Here we show that when human teratocarcinoma cells (Ntera2) are induced to differentiate into postmitotic neurons (NT2N), there is a major change in the proportion of GDI alpha relative to GDI beta, Under these conditions, certain Rab GTPases associate preferentially with either GDI alpha or GDI beta, irrespective of the relative abundance of the GDI isoform. These findings suggest that heretofore unrecognized functional specificity may exist between the two major forms of GDI.