Electron microscope autoradiography has been used to localize the glycoprotein transported between successive compartments of the Golgi in a cell-free system. Both donor and acceptor Golgi fractions contain stacks of cisternae, which remain as discrete populations even after prolonged incubations together. The glycosylated VSV G protein, having received 3H-GlcNAc residues following its transport in vitro, is entirely within the population of acceptor stacks from the uninfected wild-type Golgi population (those housing GlcNAc transferase l). Quantitation of 3H grains reveals between 6,000 and 12,000 molecules of G protein introduced into each acceptor cisterna as a result of transport in the cell-free system, amounting to approximately 5% of its total membrane protein. This represents about the full complement of transported protein normally contained in a Golgi cisterna in vivo. Transport in the cell-free system is efficient and specific, preserving the integrity of the Golgi stack and its individual cisternae.