Protease-activated receptors (PARs) are a unique family of G-coupled receptors which are activated by proteolytic cleavage of one of its extracellular domains so as to unmask and create a new N-terminal sequence. Subsequently, it acts as a tethered ligand which can bind to an adjacent extracellular domain of the receptor, resulting in intracellular signaling followed by internalization of the receptor [1]. To date, four PAR subtypes have been identified: PAR-1, PAR-2, PAR-3 and PAR-4. PAR-2 is increasingly recognized to have a pro-inflammatory role, with previous work by our group identifying a key role for this receptor in murine chronic arthritis [2] and in the rheumatoid synovium [3]. There is now emerging evidence suggesting a pro-inflammatory role for PAR-4 [4]. The aim of this study was to compare for the first time the differential expression of all the PAR mRNAs in unstimulated fibroblast-like synoviocytes (FLS) cultured from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Synovial samples were obtained with informed consent from 3 OA, and 3 RA patients, and were incubated under standard conditions to obtain cultures of FLS. Total cellular RNA was extracted from FLS using TRIzol Reagent (Life Technologies, UK). To exclude genomic DNA contamination, all RNA samples were treated with DNase1 (Invitrogen) as per the manufacturer’s protocol. RT-PCR was performed using the Invitrogen Reverse Transcription System. Aliquots of cDNA were used as templates in PCR using Megamix (Microzone, UK) with the following primer pairs: PAR-1, 5’-TGT GAA CTG ATC ATG TTT ATG -3’and 5’-TTC GTA AGA TAA GAG ATA TGT-3’, (708bp); PAR-2, 5’-GAA GCC TTA TTG GTA AGG TTG-3’and 5’-AAC ATC ATG ACA GGT CGT GAT-3’, (580bp); PAR-3, 5’-GAA AGC CCT CAT CTT TGC AG-3’ and 5’-AGG TGA AAG GAT GGA CGA TG-3’, (599bp); and PAR-4, 5’-GGC AAC CTC TAT GGT GCC TA-3’ and 5’-TTC GAC CCA GTA CAG CCT TC-3’ (243bp) .The signal yielded by the PAR primer pairs was normalized to GAPDH. Thermal cycling was performed under the conditions for PCR detection of PAR-2 [2]. PCR products were separated on a 1.5% agarose gel and visualised by ethidium bromide staining. RT-PCR analysis consistently revealed the expression of both PAR-2 and PAR-4 mRNA in unstimulated FLS of all 3 OA and 3 RA samples, whilst no PAR-1 or PAR-3 mRNA was detected in any of the samples. None of the PAR mRNAs were detectable with the omission of reverse transcriptase, confirming absence of genomic DNA contamination. These findings suggest co-expression of PAR-2 and PAR-4 mRNA may independently or interactively contribute to the development of synovitis.