Cell substrate adhesion is a prerequisite for invasion and the subsequent formation of metastases. Therefore, we designed monoclonal antibodies (MAbs) against epitopes on the extracellular cell membrane domain of SK-BR-3 cells. One of the antibodies, called MAb 14C5, binds to an extracellular epitope of a plasma membrane antigen of SK-BR-3 and MCF-7 human breast cancer cells. This MAb 14C5 is able to inhibit cell substrate adhesion, not only on culture-treated plastic but also on host tissue, and therefore prevents invasion and metastases. We evaluated the tissue distribution of the 14C5 antigen by immunohistochemistry. The antigen is specifically overexpressed in 64% of invasive ductal adenocarcinomas of the breast (n = 33), in all investigated cases of invasive squamous cell carcinoma (n = 7) and in 40% of basocellular carcinomas of the skin (n = 5). The 14C5 molecule is located on the cell membrane of the carcinoma cells. However, when the tumor is characterized by a highly invasive phenotype, 65% of the cases also show an extensive stromal expression on the fibroblasts between the tumor cells (n = 71). This stromal expression is caused by the presence of the 14C5 antigen on the membrane of the adjacent fibroblasts. In normal tissues as well as in the stroma surrounding in situ carcinomas of the breast (n = 15), no expression of the 14C5 antigen occurred. A 90-kDa protein was purified from lysates of human breast cancer cells using a 14C5 MAb Sepharose column and is considered as the antigen recognized by the MAb 14C5.