An N-ethylmaleimide-sensitive transport component (NSF) has been purified on the basis of its ability to support transport between Golgi cisternae. We now report that NSF is needed for membrane fusion. Thus, when NSF is withheld from incubations of Golgi stacks with cytosol and ATP, uncoated transport vesicles accumulate. Biochemical experiments confirm this conclusion and reveal that NSF is needed to form the first of two previously described prefusion complexes. NSF, therefore, acts within a cascade in which a vesicle-cisterna complex is matured until it is competent for fusion. We suggest that this reflects the stepwise assembly of a multisubunit "fusion machine" following vesicle attachment.