Gene expression analysis has become a promising tool in predicting the clinical course of malignant disease and the response to antineoplastic therapy. Surprisingly, only little is known about the protein expression pattern of human tumors. Recent advances in proteomic analysis allow proteins of interest to be identified by their expression and/or modification pattern in 2-DE rather than using the traditional approach of translating gene expression data. To identify a proteomic pattern that is characteristic for malignant breast epithelium, we performed differential 2-DE analysis in sets of microdissected malignant breast epithelia and corresponding adjacent normal breast epithelia from five patients with invasive breast carcinoma. Thirty-two protein spots were found to be selectively regulated in malignant epithelium, and were subjected to MALDI-TOF and/or immunoblotting for protein identification. Thirteen of the identified proteins had previously not been associated with breast cancer. The validity of these findings was confirmed by literature review and immunohistochemistry for identified proteins in an independent cohort of 50 breast cancer specimens. We here describe, for the first time, a proteomic analysis of matched normal and malignant epithelia from invasive breast carcinomas. This strategy leads to a better understanding of oncogenesis at an operational level and helps to characterize the malignant phenotype of individual tumors, and thereby to identify novel targets for antineoplastic therapy.