Abstract Summary COPII mediated ER-to-golgi membrane traffic was first reconstituted using crude cell extracts thirty years ago. Twenty years ago, the formation of COPII vesicles was recapitulated using pure proteins and lipids. However, the consumption of COPII vesicles – their uncoating, tethering, docking, and fusion with accep- tor compartments, is still poorly understood. This is in large measure because the crude transport assay developed thirty years ago has not been supplanted by a system employing chemically defined components. In this Project we introduce such a system and use it to test hypotheses about the sequence of biochemical subreactions in COPII vesicle traffic, the components that execute these subreactions, and the mechanisms through which they function.