Proteinase-activated receptor-2 (PAR-2) is a G protein–coupled receptor that is cleaved by trypsin within the NH₂-terminus, exposing a tethered ligand that binds and activates the receptor. We examined the secretory effects of trypsin, mediated through PAR-2, on well-differentiated nontransformed dog pancreatic duct epithelial cells (PDEC). Trypsin and activating peptide (AP or SLIGRL-NH₂, corresponding to the PAR-2 tethered ligand) stimulated both an ¹²⁵I^– efflux inhibited by Ca²⁺-activated Cl^– channel inhibitors and a ⁸⁶Rb⁺ efflux inhibited by a Ca²⁺-activated K⁺ channel inhibitor. The reverse peptide (LRGILS-NH₂) and inhibited trypsin were inactive. Thrombin had no effect, suggesting absence of PAR-1, PAR-3, or PAR-4. In Ussing chambers, trypsin and AP stimulated a short-circuit current from the basolateral, but not apical, surface of PDEC monolayers. In monolayers permeabilized basolaterally or apically with nystatin, AP activated apical Cl^– and basolateral K⁺ conductances. PAR-2 agonists increased [Ca²⁺]_i in PDEC, and the calcium chelator BAPTA inhibited the secretory effects of AP. PAR-2 expression on dog pancreatic ducts and PDEC was verified by immunofluorescence. Thus, trypsin interacts with basolateral PAR-2 to increase [Ca²⁺]_i and activate ion channels in PDEC. In pancreatitis, when trypsinogen is prematurely activated, PAR-2–mediated ductal secretion may promote clearance of toxins and debris.