Using a cell-free reconstitution system, we have characterized a cytosol- and ATP-dependent process that leads to the formation of membrane tubules from isolated Golgi complexes. These membrane tubules are uniform in diameter (50-70 nm) and morphologically identical to ones normally seen in cells and to those which are enhanced following brefeldin A treatment. Tubulation was strictly dependent on an activity present in an organelle-free extract of bovine brain cytosol and hydrolyzable ATP. Tubule formation was saturable with respect to both cytosol and ATP with half-maximal induction occurring at approximately 0.5 mg/mL cytosol and 10-20 microM ATP. Mild proteolytic treatment of Golgi membranes significantly reduced the extent of tubulation to subsequently added cytosol, suggesting that the tubulation activity interacts with Golgi-associated membrane proteins. The cytosolic tubulation activity was heat-labile, nondialyzable, and precipitated in ammonium sulfate. This activity could be followed through various chromatographic steps to yield fractions enriched in a major 40 kDa protein and several other minor proteins of approximately 80, 60, and 30 kDa. Monospecific antibodies against the 40K protein inhibited the cytosol-dependent tubulation of Golgi membranes in the cell-free system. Gel filtration chromatography suggests that the tubulation activity has a native molecular weight of approximately 125,000-140,000. These results establish the existence of cytosolic protein factors that regulate the formation of Golgi membrane tubules, and will provide the means for a biochemical dissection of membrane tubulation.