Trypsin and mast cell tryptase cleave within the extracellular N terminus of proteinase-activated receptor-2 (PAR-2), exposing a tethered ligand (SLIGRL) that binds and activates the cleaved receptor. We examined the neuronal expression of PAR-2 and its role in intestinal ion transport. Short-circuit current elevations in response to trypsin or the receptor-activating peptide SLIGRL-NH₂ were measured in sheets of mucosa-submucosa from porcine ileum. SLIGRL-NH₂ or trypsin rapidly elevated short-circuit current after their contraluminal application with respective 50% effective concentrations of 184 and 769 nM. Their actions were attenuated after contraluminal administration of the neuronal conduction blocker saxitoxin (0.1 μM); the cyclooxygenase inhibitor indomethacin (10 μM); or the Na⁺/K⁺/Cl⁻ cotransport inhibitor furosemide (10 μM), but not by atropine (0.1 μM), a muscarinic cholinergic antagonist. In addition, soybean trypsin inhibitor (5 μg/ml) reduced mucosal responses to trypsin. The δ-opioid agonist [d-Pen^2,5]-enkephalin (0.1 μM) inhibited trypsin action, an effect that was prevented by naltrindole (0.1 μM), a δ-opioid antagonist. PAR-2 immunofluorescence was localized in the mucosa using a receptor-specific antibody. PAR-2-like immunoreactivity was detected in myenteric and submucosal neurons, nerve fibers innervating ileal smooth muscle and mucosa, and in enteroendocrine cells. Some neurons coexpressed PAR-2- and choline acetyltransferase-like immunoreactivity. These results indicate that PAR-2 is expressed on cholinergic and noncholinergic submucosal neurons in porcine ileum. PAR-2 agonists stimulate active anion secretion by a neurogenic mechanism that is modulated by prostanoids and opioids. These receptors may have a potentially important role in intestinal neuroimmunomodulation.