A monoclonal antibody ( 53FC3 ) has been produced against a Golgi membrane protein with a mol. wt. of 135 000 which was originally identified using a polyclonal antiserum. Treatment of isolated, intact Golgi vesicles with protease caused a decrease in mol. wt. of 5000-10 000, whereas in the presence of Triton X-100, the protein was completely degraded. This shows that the protein spans the bilayer and that most of its mass is on the luminal side of Golgi membranes. Using two immunoelectron microscopic techniques, the protein was found in one or two cisternae on one side of the Golgi stack which, in normal rat kidney cells, had 4-6 cisternae. As an illustration of the use to which this monoclonal antibody can be put we present a light microscopic study of the disassembly and reassembly of the Golgi complex during mitosis.