The specificity of vesicular transport in a cell is determined by the formation of vesicles with specific contents from a donor compartment and their selective fusion with the appropriate acceptor compartment. Several of the latter fusion steps have been investigated in detail using cell-free systems, and work with these systems as well as genetic evidence has revealed a role for GTP-binding proteins in membrane fusion processes. We have reconstituted the formation of constitutive secretory vesicles and immature secretory granules from the trans Golgi network in a cell-free system. We show here that the budding of both types of post-Golgi vesicles is inhibited by non-hydrolysable analogues of GTP, which suggests a more widespread role for GTP-binding proteins in membrane traffic than previously assumed.