17beta-Estradiol (E(2)) is a key regulatory steroid hormone that is involved in the control of a number of developmental and other functions. The aim of the present work was to identify estrogen-dependent proteomic changes by determining the levels of expressed proteins in MCF-7 human breast cancer cells following treatment with E(2). A number of methods exist for differential analysis of complex proteomic mixtures. Here, a label-free mass spectrometric approach comparing the ion intensities of tryptic peptides was adopted, which was combined with prefractionation of whole cell lysate proteins by 1-D SDS-PAGE. Using this approach, 60 proteins were found to be affected by E(2). These comprised 55 up-regulated and five down-regulated proteins. These proteins varied widely in their physiochemical properties with pIs of 4-12 and molecular weights of 9-500 kDa. Pathway analysis revealed that the majority of changes were related and together describe an up-regulated pathway consistent with the events of cell proliferation. The quantitative approach used here is relatively straightforward, avoids the use of costly labelling reagents, was reproducible within acceptable limits and has a linear response over a useful concentration range.