Matriptase, a trypsin-like serine protease with two potential regulatory modules (low density lipoprotein receptor and complement C1r/s domains), was initially purified from T-47D breast cancer cells. Given its plasma membrane localization, extracellular matrix-degrading activity, and expression by breast cancer cells, this protease may be involved in multiple aspects of breast tumor progression, including cancer invasion. In breast cancer cells, matriptase was detected mainly as an uncomplexed form; however, low levels of matriptase were detected in complexes. In striking contrast, only the complexed matriptase was detected in human milk. The complexed matriptase has now been purified. Amino acid sequences obtained from the matriptase-associated proteins reveal that they are fragments of a Kunitz-type serine protease inhibitor that was previously reported to be an inhibitor of the hepatocyte growth factor activator. In addition, matriptase and its complexes were detected in milk-derived, SV40 T-antigen-immortalized mammary luminal epithelial cell lines, but not in human foreskin fibroblasts or in HT-1080 fibrosarcoma cells. These results suggest that the milk-derived matriptase complexes are likely to be produced by the epithelial components of the lactating mammary gland in vivo and that the activity and function of matriptase may be differentially regulated by its cognate inhibitor, comparing breast cancer with the lactating mammary gland.