To attain a comprehensive membrane proteome of two strains of Corynebacterium glutamicum (l-lysine-producing and the characterized model strains), both sample pretreatment and analysis methods were optimized. Isolated bacterial membranes were digested with trypsin/cyanogen bromide or trypsin/chymotrypsin, and a complementary protein set was identified using the multidimensional protein identification technology (MudPIT). Besides a distinct number of cytosolic or membrane-associated proteins, the combined data analysis from both digests yielded 326 integral membrane proteins ( approximately 50% of all predicted) covering membrane proteins both with small and large numbers of transmembrane helices. Also membrane proteins with a high GRAVY score were identified, and basic and acidic membrane proteins were evenly represented. A significant increase in hydrophobic peptides with distinctly higher sequence coverage of transmembrane regions was achieved by trypsin/chymotrypsin digestion in an organic solvent. The percentage of identified membrane proteins increased with protein size, yielding 80% of all membrane proteins above 60 kDa. Most prominently, almost all constituents of the respiratory chain and a high number of ATP-binding cassette transport systems were identified. This newly developed protocol is suitable for the quantitative comparison of membrane proteomes and will be especially useful for applications such as monitoring protein expression under different growth and fermentation conditions in bacteria such as C. glutamicum. Moreover with more than 50% coverage of all predicted membrane proteins (including the non-expressed species) this improved method has the potential for a close-to-complete coverage of membrane proteomes in general.