The increased cell surface expression of the serine integral membrane protease, seprase, has been associated with the invasive behavior of human melanoma cell lines in vitro. The present study investigates the expression of seprase in malignant, premalignant, benign, and normal human breast tissues. The 170-kDa gelatinase activity of seprase was identified in extracts of infiltrating ductal carcinomas (IDC). Protein bands corresponding to the proteolytically active 170-kDa seprase dimer and its 97-kDa seprase subunit protein were identified by immunoblot analysis of IDC extracts using an antiserum elicited against immunoaffinity-purified seprase. Immunohistochemical analysis of seprase expression in 41 formalin-fixed and paraffin-embedded specimens of human breast tissue revealed preferential immunoreactivity with the malignant cells of IDC (27 cases). Within individual IDC specimens, the stromal cells or morphologically normal epithelium revealed low labeling that was always significantly less than the labeling of neoplastic cells. Lymph node metastases of IDC cells were also strongly positive, but the lymphoid tissue in affected nodes was not stained. Neoplastic cells in DC in situ (5 cases) exhibited variable levels of staining. Epithelial cells of benign fibroadenoma specimens (2 cases) and benign proliferative breast disease (5 cases) exhibited little or no immunoreactivity. Epithelial cells of normal breast tissue (1 case) were not stained. The overexpression of seprase by DC cells is consistent with seprase having a role in facilitating invasion and metastasis of IDC of the breast. The cell surface localization of seprase could be used to target therapeutic agents to malignant breast cells.