Membrane proteins play a central role in the interaction of the cell with its environment and in the function of subcellular organelles. The current study focused on developing a better understanding of the membrane proteome of two well-characterized breast cancer cell lines. Membranes from osmotically lysed BT474 and MCF7 cells were treated with cyanogen bromide followed by a combination of trypsin and Staphylococcus V8 protease to obtain hydrophilic peptides from membrane proteins. The complex peptide mixtures obtained were separated by 2-dimensional liquid chromatography coupled online with a nano-electrospray ionization ion trap mass spectrometer (2D LC/nanoESI-MS). The strong cation exchange column used in the first dimension of the separation was eluted in an automated fashion using a series of salt steps of increasing concentration. Peptides eluted from each of the salt steps were separated using a capillary reversed-phase HPLC column, the output of which was directed through a nano-electrospray fused silica tip into the mass spectrometer. Peptides were fragmented by collision-induced dissociation (CID) and analyzed by data-dependent MS/MS followed by database searching using the Sequest algorithm. Analysis of the data revealed both similarities and expected differences between proteins identified from these cell lines. As demonstrated by others, mRNA and the HER2/neu protein tyrosine kinase-linked receptor in BT474 cells is up regulated compared to its level in MCF7, while the expression of the estrogen receptor alpha is known to be up regulated in MCF7 cells. As expected, our studies showed identification of peptides from HER2 in BT474 while estrogen receptor peptides were detected in the MCF7 line. A total of 604 proteins were identified from BT474 membranes while 313 proteins were found from MCF7. The results are discussed in terms of the known differences in both protein and mRNA expression between these two breast cancer cell lines and also in the context of other known phenotypic differences between these cells.