The effects of PAR₂-activating PAR₂-activating peptides, SLIGRL (SL)-NH₂, and trans-cinnamoyl-LIGRLO (tc)-NH₂ were compared with the action of trypsin, thrombin, and the PAR₁ selective-activating peptide: Ala-parafluoroPhe-Arg-cyclohexylAla-Citrulline-Tyr (Cit)-NH₂ for stimulating intestinal ion transport. These agonists were added to the serosa of stripped rat jejunum segments mounted in Ussing chambers, and short circuit current (Isc) was used to monitor active ion transport. The relative potencies of these agonists also were evaluated in two bioassays specific for the activation of rat PAR₂: a cloned rat PAR₂ cell calcium-signaling assay (PAR₂-KNRK cells) and an aorta ring relaxation (AR) assay. In the Isc assay, all agonists, except thrombin, induced an Isc increase. The SL-NH₂-induced Isc changes were blocked by indomethacin but not by tetrodotoxin. The relative potencies of the agonists in the Isc assay (trypsin≫SL-NH₂>tc-NH₂>Cit-NH₂) were strikingly different from their relative potencies in the cloned PAR₂-KNRK cell calcium assay (trypsin≫>tc-NH₂ ≅ SL-NH₂≫>Cit-NH₂) and in the AR assay (trypsin≫>tc-NH₂ ≅ SL-NH₂). Furthermore, all agonists were maximally active in the PAR₂-KNRK cell and AR assays at concentrations that were one (PAR₂ -activating peptides) or two (trypsin) orders of magnitude lower than those required to activate intestinal transport. Based on the distinct potency profile for these agonists and the considerable differences in the concentration ranges required to induce an Isc effect in the intestinal assay compared with the PAR₂-KNRK and AR assays, we conclude that a proteinase-activated receptor, pharmacologically distinct from PAR₂ and PAR₁, is present in rat jejunum and regulates intestinal transport via a prostanoid-mediated mechanism.