Transport of the vesicular stomatitis virus (VSV)-encoded glycoprotein (G protein) between successive compartments of the Golgi in a cell-free system is measured by the coupled incorporation of N-[3H]acetylglucosamine (GlcNAc). This glycosylation occurs when G protein is transported from a "donor" compartment in Golgi membranes that lack GlcNAc transferase I (from VSV-infected CHO clone 15B cells) to the next "acceptor" compartment in a Golgi population from wild-type CHO cells (containing the GlcNAc transferase but not G protein). Here we present a detailed characterization of the conditions required to achieve transport in vitro. We find that donor and acceptor activities differ markedly in certain of their properties. The donor activity is inhibited by N-ethylmaleimide but the acceptor activity is resistant. Donor activity is unstable in the absence of ATP or the cytosol fraction; acceptor activity is much more stable. This asymmetry may reflect the vectorial nature of the underlying biochemistry of protein transport. Both donor and acceptor are trypsin-sensitive, implying a need for cytoplasmically oriented membrane proteins. Transport occurs only in a restricted range of close to physiological conditions. ATP is absolutely required, although as little as 1 microM is sufficient. Transport is inhibited by ATP-gamma-sulfate and vanadate, suggesting that ATP hydrolysis is needed. By contrast, ionophores that dissipate membrane potentials and proton gradients do not inhibit transport. Monensin was also without effect in the cell-free system.