PARs are a novel class of GPCRs activated by proteolytic unmasking of a tethered ligand domain within the N-terminus which in turn initiates receptor activation. PAR2, a trypsin and tryptase activated member, has been implicated in cardiovascular disease (Coelho et al., 2003; McGuire, 2004). Previous studies reported that activation of hPAR2 by mast cell tryptase can be regulated by the post-translational modification N-linked glycosylation (Compton et al., 2002). Since palmitoylation is an important post-translational modification that can have profound effects on GPCR function, we investigated the role of the putative palmitoylation site (C361) within hPAR2. Wild-type hPAR2 (wt-hPAR2) and mutant hPAR2C361A were permanently expressed in CHO cells. Intracellular calcium signalling was used to compare the functional activity between wt-hPAR2 and hPAR2C361A cell lines with matched receptor expression. hPAR2C361A showed a significant reduction in sensitivity towards both trypsin and the selective hPAR2 activating peptide SLIGKV-NH2 (4-fold and 6-fold respectively). In addition, hPAR2C361A displayed a marked decrease in maximal response to both agonists compared to wt-PAR2. ERK1/2 activation was assessed by western blot analysis of cell lysates from agonist treated wt-hPAR2 and hPAR2C361A cell lines. For wt-hPAR2, trypsin and SLIGKV-NH2 stimulated a modest and transient (5min) increase in phosphorylated-ERK1/2 (p-ERK1/2) which declined thereafter to baseline levels. In contrast, for hPAR2C361A trypsin and SLIGKV-NH2 stimulated a robust increase in p-ERK1/2 that remained elevated for up to 20min before declining to baseline. These findings suggest that hPAR2C361 and potentially receptor palmitoylation play an important role in regulating the signalling pathways utilised by hPAR2.