The gram-positive bacterium Staphylococcus aureus is a serious human pathogen causing a wide variety of diseases, and its increasing resistance towards all available antibiotics makes its further investigation absolutely essential. We examined the membrane proteome of exponentially growing cells of S. aureus COL, since this sub-proteome plays a major role for the virulence of the bacterium in its host. In general, an analysis of membrane proteins is impeded by their hydrophobic nature, as well as by a high abundance of many cytosolic proteins. The implementation of three different technologies, 1D-gel-LC-, 2D-LC-, and a membrane shaving approach combined with MS/MS analyses, enabled an identification of 271 integral and 86 peripheral membrane proteins from exponentially growing cells. In particular, the latter approach which combined membrane shaving with a subsequent chymotrypsin digest of integral membrane domains of proteins greatly facilitated the detection of hydrophobic peptides derived from membrane spanning segments (713 peptides, 60 % of all peptides), and therefore yielded almost exclusively highly hydrophobic integral membrane proteins (96.7 %). A comparison of the various methods disclosed the 1D-gel-LC- and the shaving approach to be highly complementary techniques. A combination of them will reveal a most comprehensive view on membrane proteomes.