Proteinase-activated receptor (PAR) type 2 (PAR-2) has been shown to mediate ion secretion in cultured epithelial cells and rat jejunum. With the use of a microUssing chamber, we demonstrate the role of PAR-2 for ion transport in native human colonic mucosa obtained from 30 normal individuals and 11 cystic fibrosis (CF) patients. Trypsin induced Cl⁻ secretion when added to the basolateral but not luminal side of normal epithelia. Activation of Cl⁻ secretion by trypsin was inhibited by indomethacin and was further increased by cAMP in normal tissues but was not present in CF colon, indicating the requirement of luminal CF transmembrane conductance regulator. Effects of trypsin were largely reduced by low Cl⁻, by basolateral bumetanide, and in the presence of barium or clotrimazole, but not by tetrodotoxin. Furthermore, trypsin-induced secretion was inhibited by the Ca²⁺-ATPase inhibitor cyclopiazonic acid and in low-Ca²⁺ buffer. The effects of trypsin were almost abolished by trypsin inhibitor. Thrombin, an activator of PAR types 1, 3, and 4, had no effects on equivalent short-circuit currents. The presence of PAR-2 in human colon epithelium was confirmed by RT-PCR and additional experiments with PAR-2-activating peptide. PAR-2-mediated intestinal electrolyte secretion by release of mast cell tryptase and potentiation of PAR-2 expression by tumor necrosis factor-α may contribute to the hypersecretion observed in inflammatory processes such as chronic inflammatory bowel disease.