We tested the hypothesis that human tissue kallikreins (hKs) may regulate signal transduction by cleaving and activating proteinase-activated receptors (PARs). We found that hK5, 6 and 14 cleaved PAR N-terminal peptide sequences representing the cleavage/activation motifs of human PAR₁ and PAR₂ to yield receptor-activating peptides. hK5, 6 and 14 activated calcium signalling in rat PAR₂-expressing (but not background) KNRK cells. Calcium signalling in HEK cells co-expressing human PAR₁ and PAR₂ was also triggered by hK14 (via PAR₁ and PAR₂) and hK6 (via PAR₂). In isolated rat platelets that do not express PAR₁, but signal via PAR₄, hK14 also activated PAR-dependent calcium signalling responses and triggered aggregation. The aggregation response elicited by hK14 was in contrast to the lack of aggregation triggered by hK5 and 6. hK14 also caused vasorelaxation in a phenylephrine-preconstricted rat aorta ring assay and triggered oedema in an in vivo model of murine paw inflammation. We propose that, like thrombin and trypsin, the kallikreins must now be considered as important ‘hormonal’ regulators of tissue function, very likely acting in part via PARs.