We describe the potential role of ADP-ribosylation factor (ARF) in vesicular trafficking using an in vitro assay that efficiently reconstitutes transport between the endoplasmic reticulum (ER) and the cis-Golgi compartment in mammalian semi-intact cells, a population of cells in which the plasma membrane is physically perforated to reveal intact ER and Golgi compartments. We demonstrate that peptides identical to the amino-terminal domain of ARF, which inhibit ARF cofactor activity in cholera toxin-catalyzed ADP-ribosylation of G alpha S (Kahn, R. A., Randazzo, P., Serafini, T., Weiss, O., Rulka, C., Clark, J., Amherdt, M., Roller, P., Orci, L., and Rothman, J. E. (1992) J. Biol. Chem. 267, 13039-13046), inhibit transport of the vesicular stomatitis virus G protein between the ER and cis-Golgi compartment. Inhibition of transport was rapid (t1/2 = 30-60 s) and irreversible. Half-maximal inhibition was observed at concentrations of 15 and 22 microM with peptides identical to the amino-terminal domain of the human ARF4 (hARF4) protein and the human ARF1 protein, respectively. Kinetic analysis of vesicular stomatitis virus G protein transport suggested that the hARF4 peptide inhibits a late vesicle fusion step. In addition, incubation of semi-intact cells in the presence of the myristoylated form human ARF1 (hARF1myr) protein, but not the nonmyristoylated form of ARF1, inhibited transport. In contrast to peptide, the hARF1myr blocked an early transport step, similar to that observed with guanosine 5'-3-O-(thio)triphosphate. These results suggest that ARF and components facilitating ARF function play an important role in the cyclical fission and fusion of transport vesicles mediating ER to Golgi trafficking.