Integral membrane proteins, particularly those with more than one transmembrane domain, have traditionally been difficult to separate by two-dimensional electrophoretic methods. Here we report the adaptation of a previously published procedure [D. E. Macfarlane (1989) Anal. Biochem. 176, 457-463] for the analytical and semipreparative separation of membrane proteins. The first dimension involves discontinuous gel electrophoresis in an acidic buffer system using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC). The second dimension consists of discontinuous SDS-PAGE. Using carbonate-washed membranes of synaptic vesicles and clathrin-coated vesicles as examples, we demonstrate that complex membrane protein mixtures can be resolved with a resolution at least fivefold higher than that of one-dimensional SDS-PAGE. Protein patterns are highly reproducible and proteins with single or multiple transmembrane domains are resolved as clearly distinct spots. Smearing of bands or losses of protein are minimal. Several spots were identified by immunoblotting and internal sequencing. Thus, this method is suitable for the analytical and semipreparative characterization of membrane proteins derived from complex biological samples.