Seprase or Fibroblast activation protein (FAP) is an integral membrane serine peptidase, which has been shown to have gelatinase activity. It appears to act as a proteolytically active 170-kDa dimer, consisting of two 97-kDa subunits. It is a member of the group type II integral serine proteases, which include dipeptidyl peptidase IV (DPPIV/CD26) and related type II trans-membrane prolyl serine peptidases, which exert their mechanisms of action on the cell surface. DPPIV and Seprase exhibit multiple functions due to their abilities to form complexes with each other and to interact with other membrane-associated molecules. Localization of these protease complexes at cell surface protrusions, called invadopodia, may have a prominent role in processing soluble factors and in the degradation of extracellular matrix components that are essential to the cellular migration and matrix invasion that occur during tumour invasion, metastasis and angiogenesis. Seprase was isolated and purified from bovine serum, yielding a specific activity of 166.41 units/mg. The purified soluble Seprase was shown to have both exopeptidase and endopeptidase activity. The soluble form of the glycoprotein was shown to bind to the Wheat Germ Agglutinin (WGA)-Lectin Affinity Chromatography. Biochemical studies show that Seprase has a pH optimum of 8.0 and it is thermostable (up to 40 °C). The second order rate constant, k2 for DFP inhibition of Seprase was determined to be 3.31 x 103 Af V 7. Positional scanning of the Pi dipeptide library determined that Seprase has a marked preference for proline in the Si subsite. Data obtained during screening o f the P2 sub-library revealed a much broader specificity in the S2 binding pocket of bovine Seprase. In the S2 subsites, this study shows that Seprase has a preference for Nor leucine, Alanine, Leucine, Glycine, Arginine, Methionine and does not tolerate aromatic, strongly basic or acidic residues. The kinetic constant Km determined for purified Seprase was 82.10¡jM. Seprase was found to have an average kCat / Km ratio of 1.17 x 105 M 1 s'1 for cleavage o f the fluorimetric substrate Z-Gly- Pro-AMC. Tissue localisation studies identified Seprase activity in bovine large intestine, serum, kidney, liver and spleen and also in the breast cancer cell line Hs578T. Seprase was successfully cloned into prokaryotic and eukaryotic expression systems. Clinical studies on serum samples from breast cancer patients indicated that Seprase levels are elevated in patients with invasive ductal carcinoma.