The function of the guanosine triphosphate (GTP)-binding protein Ypt1 in regulating vesicular traffic was studied in a cell-free system that reconstitutes transport from the endoplasmic reticulum to the Golgi. Blocking the Ypt1 protein activity resulted in accumulation of vesicles that act as an intermediate passing between the two compartments. The Ypt1 protein was found on the outer side of these vesicles. The transport process is completed by fusion of these vesicles with the acceptor compartment, and Ypt1 protein activity was needed for this step. Thus, a specific GTP-binding protein is required for either attachment or fusion (or both) of secretory vesicles with the acceptor compartment during protein secretion.