Examination of a cell-free reconstitution of intercompartmental transport through the Golgi apparatus has enabled detection of two intermediates in the pathway (Balch, W. E., Glick, B. S., and Rothman, J. E. (1984) Cell 39, 525-536). These intermediates are thought to represent stages in the budding and fusion reactions of transport vesicles mediating such a transport process. Here we describe a new transport intermediate that is interposed between the previously established primed donor formation and the N-ethylmaleimide (NEM)-resistant acceptor intermediates. Consumption of this intermediate requires much less cytosol than its formation, and thus it has been termed the "dilution-resistant" intermediate. The dilution-resistant intermediate only forms in the presence of donor and acceptor membranes, and its consumption is sensitive to NEM. The transition from this state to the later, NEM-resistant form of the prefusion complex requires ATP as well as cytosol and may represent a processing of transport vesicles to permit their fusion.