Transport of the VSV-encoded glycoprotein (G protein) between successive compartments of the Golgi has been reconstituted in a cell-free system and is measured, in a rapid and sensitive new assay, by the coupled incorporation of 3H-N-acetylglucosamine (GlcNAc). This glycosylation occurs when G protein is transported during mixed incubations from the "donor" compartment in Golgi from VSV-infected CHO clone 15B cells (missing a key Golgi GlcNAc transferase) to the next, successive "acceptor" compartment (containing the GlcNAc transferase) in Golgi from wild-type CHO cells. Golgi fractions used in this assay have been extensively purified, and account for all of the donor and acceptor activity in the cells. Together with several other lines of evidence, this indicates that the cell-free system is highly specific, measuring only transport between sequential compartments in the Golgi stack. Transport in vitro is almost as efficient as in the cell, and requires ATP and the cytosol fraction in addition to protein components on the cytoplasmic surface of the Golgi membranes.