Proliferation and differentiation of mammary epithelial cells are governed by hormonal stimuli, cell-cell and cell-matrix interactions. Terminal differentiation of mammary epithelial cells depends upon the action of the lactogenic hormones, insulin, gluco corticoids and prolactin, that enable them to synthesise and secrete milk proteins. These differentiated cells are polarized and carry out vectorial transport of milk constituents across the apical plasma membrane. To gain additional insights into the me c hanisms governing differentiation of mammary epithelial cells, we identified proteins whose expression distinguishes proliferating from differentiated mammary epithelial cells. For this purpose we made use of the HC11 mammary epithelial line, which is cap able of differentiation in response to lactogenic hormones. Using two dimensional gel electrophoresis and mass spectrometry, we found about 60 proteins whose expression levels changed in between these two differentiation states. Bioinformatic analysis rev ealed differential expression of cytoskeletal components, molecular chaperones and regulators of protein folding and stability, calcium binding proteins and components of RNA processing pathways. The actin cytoskeleton is asymmetrically distributed in dif ferentiated epithelial cells and the identification of proteins involved in mRNA binding and localization suggests that asymmetry might in part be achieved by controlling cellular localization of mRNAs. The proteins identified provide insights into the differentiation of mammary epithelial cells and the regulation of this process.