Human kallikrein 5 (hK5; encoded by the KLK5 gene) is a novel serine protease, predicted to have trypsin-like activity. Given that hK5 is differentially expressed in cancer and members of the human kallikrein family require cleavage of their propeptides by a trypsin-like serine protease, we hypothesized that hK5 may be implicated in tumour progression, and be a member of an enzymatic cascade pathway. In this study, recombinant hK5 was produced and shown to have trypsin-like activity, with preference or Arg over Lys for the P1 position. Its activity was inhibited by alpha 2-antiplasmin, antithrombin, alpha2-macroglobulin, SPINK5, and zinc. Extracellular matrix components, along with plasminogen, vitronectin, kininogen, fibrinogen, insulin-like growth factor binding proteins and semenogelins, were identified as putative physiological substrates of hK5. Finally, hK5 was able to activate and inactivate prohK2 and prohK3. We conclude that hK5 may be involved in an enzymatic cascade pathway and in tumour progression.