Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs). Although thrombin is a recognized physiological activator of PARs 1 and 4, the endogenous enzymes responsible for activating PAR2 in settings other than the gastrointestinal system, where trypsin can activate PAR2, are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulate PAR signalling using: (1) an HPLC-mass spectral analysis of the cleavage products yielded upon incubation of hKs 5, 6 and 14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PARs 1, 2 and 4, (2) PAR-dependent calcium signalling responses in cells expressing PARs 1, 2 and 4, and in human platelets, (3) a vascular ring vasorelaxation assay and (4) a PAR4-dependent rat and human platelet aggregation assay. We found that hKs 5, 6 and 14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/dis-arming. Further, hK14 was able to activate PARs 1, 2 and 4 and to dis-arm/inhibit PAR1. Whilst hKs 5 and 6 were also able to activate PAR2, they failed to cause PAR4-dependent aggregation of rat and human platelets, whereas hK14 did. Further, the relative potencies and maximum effects of hKs 14 and 6 to activate PAR2-mediated calcium signalling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PARs 1, 2 and 4.