A cloned rat proteinase-activated receptor (PAR)₂-expressing cell line (KNRK-rPAR₂) was used to study the structure-activity relationships (elevated intracellular Ca²⁺) for a series of: 1) PAR₁-derived receptor-activating ligands (PAR₁-APs) [SFLLR (P5), SFLLR-NH₂(P5-NH₂), SFLLRNP (P7), SFLLRNP-NH₂(P7-NH₂), and TFLLR-NH₂ (TF-NH₂)] and 2) PAR₂-derived-activating-peptides (PAR₂-APs) [SLIGRL-NH₂ (SL-NH₂), SLIGR-NH₂ (GR-NH₂), and SLIGKV-NH₂(KV-NH₂)]. The activities of the PAR-APs were compared with the PAR₂-AP analogtrans-cinnamoyl-Leu-Ile-Gly-Arg-Leu-Orn-NH₂tc-NH₂), which as a [³H]propionyl derivative ([³H]propionyl-tc-NH₂) was used to develop a radioligand-binding assay for PAR₂. The relative potencies of the PAR-APs in the Ca²⁺-signaling assay were tc-NH₂ = SL-NH₂ > KV-NH₂≅ P5-NH₂ > GR-NH₂ > P7-NH₂ > P7 > P5 > TF-NH₂. The reverse sequence PAR-APs, LSIGRL-NH₂(LS-NH₂), LRGILS-NH₂ (LR-NH₂), FSLLRY-NH₂ (FSY-NH₂), and FSLLR-NH₂(FS-NH₂), as well as the XenopusPAR₁-AP TFRIFD-NH₂, were inactive. The relative biological potencies of the peptides were in accord with their ability to compete for the binding of [³H]propionyl-tc-NH₂ (tc-NH₂= SL-NH₂ > GR-NH₂ ≅ P5-NH₂ > P5) to KNRK-rPAR₂ cells, whereas inactive peptides (FS-NH₂; LR-NH₂) showed no appreciable binding competition. Our data therefore validate a ligand-binding assay for the use in studies of PAR₂ and indicate that the relative biological potencies of the PAR₁-APs for activating rat PAR₂ parallel their ability to activate human PAR₁. The relative receptor-binding activities of the PAR-APs, although in general agreement with their relative biological activities, point to differences in the intrinsic receptor-activating activities between the several PAR-APs. The binding assay we have developed should prove of use for the further study of PAR₂-ligand interactions.