AbstractHuman kallikrein 4 (hK4) is a member of the expanded family of human kallikreins, a group of 15 secreted proteases. While this protein has been associated with ovarian and prostate cancer prognosis, only limited functional information exists. Therefore, we have undertaken an investigation of its enzymatic properties regarding substrate preference, degradation of extracellular matrix proteins, and its inhibition by various inhibitors. We successfully expressed and purified active recombinant hK4 from supernatants of thePichia pastorisexpression system. This enzyme seems to cleave more efficiently after Arg compared to Lys at the P1 position and exhibits modest specificity for amino acids at positions P2 and P3. hK4 forms complexes with α1-antitrypsin, α2-antiplasmin and α2-macroglobulin. The protease mediates limited degradation of extracellular matrix proteins such as collagen I and IV, and more efficient degradation of the α-chain of fibrinogen. The cleavage of extracellular matrix proteins by hK4 suggests that this enzyme may play a role in tissue remodeling and cancer metastasis.