The urokinase-system has been implicated in tumor spread. The serine protease urokinase-type plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1) are involved in the control of extracellular turnover, cell migration, invasion, cell signalling, proliferation, apoptosis and angiogenesis leading to a variety of different responses, under both physiological and pathological conditions. uPA and PAI-1 were the first novel tumor biological factors to be validated at the highest level of evidence regarding their clinical utility in breast cancer. However, it is unclear whether it is their (relative) levels in the tumor stroma or in the tumor cells themselves that is the most relevant to patients outcome. This is the first study in which tumor cells and stromal tissue of invasive breast carcinomas were separated by laser capture microdissection followed by ELISA-based determination of the uPA, uPAR and PAI-1 levels. In addition, we localized uPA, uPAR and PAI-1 distribution in invasive breast cancer (n=30) and in ductal carcinoma in situ (DCIS, n=30) by immunohistochemistry and in situ hybridization. We have demonstrated that no significant differences between uPA, uPAR and PAI-1 levels in tumor stroma only, tumor cells only and not separated breast cancer tissue exist (p>0.05). Our results suggest that similar expression levels of these factors in both compartments and in not separated breast cancer tissue may have the same impact on the clinical behavior of breast cancer. These results are an important issue for practical use of tissue sampling. For using uPA and PAI-1 levels as prognostic and predictive factors in breast cancer the quantity of tumor stroma in the tumor tissue specimen is not relevant for assessment patients outcome. Our results were confirmed by immunohistochemistry and in situ hybridization analysis showing that in nearly all cases of invasive carcinomas and DCIS fibroblasts as well as macrophages strongly express uPA, uPAR and PAI-1. Prompted by our immunohistological results that nearly all myoepithelial cells of DCIS exhibit uPA, uPAR and PAI-1, we investigated these important host cells in detail. We have demonstrated by multimodal methods that uPAR and PAI-1 protein and mRNA is expressed in most myoepithelial cells of DCIS. Additionally, we furnish evidence that uPAR expression of myoepithelial cells are important for uPAR Vitronectin-associated cell-matrix interaction, which regulates cell adhesion and detachment. We speculate that the loss of the anti-invasive myoepithelial cell layer in DCIS may be triggered by PAI-1 and could be an early sign of subsequent tumor cell invasion.