This chapter describes the permeabilized cell system and the methods used to study the role of Rab-GDI in the reversible membrane association of Rab proteins. The advantage of using permeabilized cells is that, while the cellular organization is maintained, the intracellular membranes are accessible to biochemical manipulations. Thus, this in vitro system closely mimics the physiological conditions in intact cells. Rab proteins are associated with specific compartments along the exocytic and endocytic pathways. Membrane association requires posttranslational modification of the C-terminal cysteines by the addition of geranylgeranyl moieties. In addition, Rab proteins are also present in the cytosol. Cytosolic Rab proteins are complexed to Rab-GDI (GDP dissociation inhibitor), a factor that inhibits the dissociation of GDP and removes Rab proteins from the membrane. These biochemical properties have led to the suggestion that Rab-GDI allows Rab proteins to shuttle between the membrane and the cytosol to serve multiple rounds of vesicular traffic.