Tissue kallikreins (KLKs) are a family of secreted serine proteinases, which are up-regulated in many cancers and at sites of inflammation. Despite their widespread expression in normal and diseased tissues, the mechanisms whereby these enzymes regulate cellular function are not clear. We have recently shown (J. Biol. Chem. 281: 32095, 2006) that in vitro, kallikreins can activate proteinase activated receptors (PARs), a family of G-protein coupled cell surface receptors. These receptors have been associated with inflammation, having either an anti- or pro-inflammatory role in different pathological settings. We hypothesized that like trypsin and thrombin, kallikreins 5, 6 and 14, can trigger an inflammatory response by activating PARs. We studied the ability of kallikreins: (1) to activate PARs 1, 2 and 4 in cell culture systems (calcium signaling), (2) to cause a PAR₄-dependent platelet aggregation, as well as a vascular relaxation in both wild-type and PAR₂ null mice, (3) like trypsin, to cause oedema in a mouse model of paw inflammation in vivo. We found that with different potencies, KLKs 5, 6 and 14 can activate PAR₂, a receptor widely implicated in inflammation and cancer. However, only KLK14 was able to activate PAR₄ in isolated platelet preparations and PAR₄-expressing cells. In addition, KLK14 prevented thrombin from activating PAR₁, a receptor known to play a role in tumour metastasis and invasion. Further, when administered in vivo, KLK14 caused a paw oedema response comparable in magnitude and time course to that generated by trypsin. The oedema was accompanied by a decreased threshold of mechanical and thermal nociception. Our data demonstrate that by activating PARs 2 and 4 and by inactivating PAR₂, widely expressed kallikreins, like KLKs 5, 6 and 14, may play a role in regulating cell signaling and local inflammatory response in many pathological settings, where trypsin or thrombin are absent.