Semi-intact cells, a cell population in which the plasma membrane is perforated to expose intact intracellular organelles (Beckers, C. J. M., Keller, D. S., and Balch, W. E. (1987) Cell 50, 523-534), efficiently reconstitute vesicular trafficking of protein from the endoplasmic reticulum (ER) to the cis Golgi compartment. We now extend these studies to biochemically dissect transport of protein between the ER and the Golgi into a series of sequential intermediate steps involved in the budding and fusion of carrier vesicles. At least two broad categories of transport intermediates can be detected, those that involve early steps in transport and those involved in late, fusion-related events. Early transport steps require the transport of protein through a novel intermediate compartment in which protein accumulates at reduced temperature (15 degrees C). We demonstrate that both entry and exit from this 15 degrees C compartment can be successfully reconstituted in vitro. A late step in delivery of protein to the cis Golgi compartment requires Ca2+ (pCa7) and is coincident with a step which is sensitive to a peptide analog which blocks interaction between the Rab family of small GTP-binding proteins and a downstream effector protein(s) (Plutner, H., Schwaninger, R., Pind, S., and Balch, W. E. (1990) EMBO J. 9, 2375-2384). The combined results suggest that a single round of vesicular transport between the ER and the Golgi involves a rapid transit through N-ethylmaleimide-sensitive, guanosine 5'-(3-O-thio)triphosphate-sensitive, ATP- and cytosol-dependent step(s) involved in vesicle formation or transport to a novel intermediate compartment, followed by a regulated fusion event triggered in the presence of Ca2+ and functional components interacting with member(s) of the Rab gene family.