Abstract: Although often used in membrane proteome studies, conventional two-dimensional gel electrophoresis (2-DE) is not well suited for resolving hydrophobic proteins. Nevertheless, an alternative technique, two-dimensional BAC/SDS-PAGE (2-DB) using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (BAC) in the first and the anionic detergent SDS in the second dimension can be utilized as a powerful tool for the separation and analysis of membrane proteins. Systematic studies demonstrated the advantage of 2-DB over one-dimensional SDS-PAGE and 2-DE with regard to membrane proteomics. While in 2-DE gels, in particular proteins with more than one transmembrane domain (TMD) are underrepresented, one-dimensional SDS-PAGE lacks sufficient resolution for large scale analyses. In contrast, 2-DB enabled the identification of extremely hydrophobic proteins like cytochrome-c oxidase subunit I from S. cerevisiae with a total of 12 known TMD. Especially the application of tube gels in the first dimension as well as the recent introduction of improved buffer systems hold a great potential for future 2-DB-based membrane studies.