Protease-activated receptors are activated by protease cleavage of the N terminus, generating a tethered ligand that interacts with extracellular loop-2. Protease-activated receptor-2 (PAR-2) is a member of this family involved in inflammatory processes as adjuvant-induced arthritis is substantially attenuated in PAR-2-deficient mice compared to wild-type mice (Ferrell et al. 2003). Here we investigate PAR-2 as a therapeutic target in arthritis using a polyclonal antibody (B5) to the trypsin-binding site on PAR-2 in a model of acute arthritis. The specificity of B5 was confirmed in parallel groups, joint swelling being induced by a synthetic PAR2 agonist or the PAR-1 activating peptide, thrombin. Acute knee joint inflammation was induced by intra-articular (i.a.) injection of a 2% carrageenan and 4% kaolin (CK; 20ml) in deeply anaesthetised (O₂/N₂O/2% halothane mixture, withdrawal reflex absent) male C57BL/6J mice (n=6). In a parallel group (n=6), B5 (20ml; 1:1000 dilution) was administered by i.a. injection 5 minutes prior to CK. A further group (n=6) was pre-treated with non-immune serum. Swelling at 24 hours was expressed as % of pre-injection values. In a separate group, swelling was induced by i.a. injection (100 mg) of a synthetic PAR-2 agonist (Ferrell et al 2003), or thrombin (20 Units). In parallel groups (n=4/group) B5 was administered and joint diameter measured as above. All animals were humanely killed. Data expressed as mean ± S.E.M. and analysed by Students unpaired t test.CK alone resulted in a 26 ± 4% increase in joint diameter at 24 hours that was significantly attenuated (P < 0.002) by B5 (6 ± 2%). The CK alone response was not significantly (P=0.36) altered by non-immune serum (20 ± 4%). The PAR-2 agonist increased joint diameter (17 ± 3%) which was significantly attenuated (P=0.01) by B5 (4 ± 1%). Thrombin produced a smaller increase in diameter (7 ± 2%) that was not significantly reduced by B5 (P=0.55), arguing that the B5 effect is specific for PAR-2. The fourfold reduction of CK-induced joint swelling suggests that inhibition of PAR-2 activation is antiinflammatory. B5 is specific for PAR-2 and its inhibition of the PAR-2 agonist argues that this antibody attenuates acute joint inflammation by receptor blockade rather than by preventing generation of the tethered ligand. These results, using a pharmacological tool, confirms and extends our earlier findings, supporting the concept of PAR-2 as therapeutic target for future studies in arthritis.