1. Rat proteinase-activated receptor-2 (PAR2) variants were stably expressed in rat KNRK cells: (a) wild-type (wt) - PAR2; (b) PAR2PRR, with the extracellular loop 2 (EL-2) sequence P231E232E233mutated to PRR and (c) PAR2NET, with the EL-2 sequence, PEEV changed to NETL. Cell lines were evaluated for their sensitivity (calcium signalling) towards trypsin and the receptor-activating peptides, SLIGRL-NH2, SLIGEL-NH2, trans-cinnamoyl(tc)-LIGRLO-NH2, and SFLLR-NH2. 2. SLIGEL-NH2 exhibited low potency (1 : 200 relative to SLIGRL-NH2) in wild-type PAR2. Its activity was increased 5 fold in PAR2PRR, but it was inactive in PAR2NET. 3. In PAR2PRR, the potencies of SLIGRL-NH2, tc-LIGRLO-NH2, and SFLLR-NH2 were decreased by 80 - 100 fold. But, the potency of trypsin was decreased by only 7 fold. 4. In PAR2NET, highly homologous in EL-2 with proteinase-activated receptor-1 (PAR1), the potency of the PAR1-derived peptide, SFLLR-NH2, was reduced by 100 fold compared with wt-PAR2, whereas the potency of the PAR2-derived AP, SLIGRL-NH2 was reduced 10 fold. In contrast, the potency of trypsin in PAR2NET was almost the same as in wt-PAR2. 5. We conclude that the acidic EL-2 tripeptide, PEE, in PAR2 plays an important role in governing agonist activity. 6. The data obtained with the PEEV-->NETL mutation suggested: (a) that SLIGRL-NH2 and SFLLR-NH2 interact in a distinct manner with PAR2 and (b) that SFLLR-NH2 may interact differently with PAR2 than it does with PAR1. 7 The differential reductions in the potencies of SLIGRL-NH2, compared with trypsin in the PAR2PRR and PAR2NET cell lines point to differences between the interactions of the trypsin-revealed tethered ligand and the free receptor-activating peptide with PAR2.