GCDFP-15 (gross cystic disease fluid protein, 15 kDa) is a secretory marker of apocrine differentiation in breast carcinoma. In human breast cancer cell lines, gene expression is regulated by hormones, including androgens and prolactin. The protein is also known under different names in different body fluids such as gp17 in seminal plasma. GCDFP-15/gp17 is a ligand of CD4 and is a potent inhibitor of T-cell apoptosis induced by sequential CD4/T-cell receptor triggering. We now report that GCDFP-15/gp17 is a protease exhibiting structural properties relating it to the aspartyl proteinase superfamily. Unexpectedly, GCDFP-15/gp17 appears to be related to the retroviral members rather than to the known cellular members of this class. Site-specific mutagenesis of Asp(22) (predicted to be catalytically important for the active site) and pepstatin A inhibition confirmed that the protein is an aspartic-type protease. We also show that, among the substrates tested, GCDFP-15/gp17 is specific for fibronectin. The study of GCDFP-15/gp17-mediated proteolysis may provide a handle to understand phenomena as diverse as mammary tumor progression and fertilization.