It was the aim of this study to design mouse monoclonal antibodies (MAbs) that can inhibit the invasion of breast cancer cells in the host tissue. Therefore, MAbs were raised against epitopes on the extracellular domain of SK-BR-3 human breast cancer cells, and biological assays were performed to test the capability of the MAbs to inhibit cell substrate adhesion. MAb 14C5 bound an extracellular plasma membrane antigen of SK-BR-3 and MCF-7 human breast cancer cells and inhibited the cell substrate adhesion of these cells in vitro. The MAb delayed the adhesion of MCF-7 and SK-BR-3 cells on precultured embryonic heart fragments (PHFS). It inhibited the destruction of the PHF by MCF-7 cells and the invasion of the PHF by SK-BR-3 cells. The MAb reacted with an epitope on the cell membrane of in situ and invasive ductal carcinomas of the breast in immunohistochemistry. Poorly differentiated, highly invasive ductal carcinomas show extensive staining of long plasma membrane extensions. Normal multilayered epithelia, normal connective tissue, and tumors derived from these tissues as well as normal breast tissue were negative. From both cell lines a protein complex consisting of two subunits with molecular weight of 50 and 90 kd, respectively, was immunoprecipitated. It is concluded that the 14C5 antigen plays a role in cell substrate adhesion and subsequently also in invasion of breast cancer cells. The 14C5 MAb was able to inhibit cell substrate adhesion and invasion in vitro of breast cancer cells.