Transient receptor potential vanilloid 4 (TRPV4) is a cation channel activated by hypo–osmotic and mechanical stimuli, and the selective synthetic agonist 4α–phorbol didecanoate (4α–PDD). Hypo–osmotic stimulation produces 5’,6’–epoxyeicosatrienoic acid (5’,6’–EET), an endogenous TRPV4 agonist. TRPV4 is expressed on a subset of nociceptive neurons and may sense painful mechanical stimuli. Nociceptive neurons also express protease–activated receptor 2 (PAR2), and proteases that activate PAR2 induce thermal and mechanical hyperalgesia. PAR2–induced thermal hyperalgesia results from PKC–mediated TRPV1 sensitization. We examined the hypothesis that PAR2 activation also causes sensitization of the related TRPV4 channel. We identified mRNA encoding TRPV4 and PAR2 in a human bronchial epithelial cell line (16HBE140–) and rat dorsal root ganglia neurons by RT–PCR. We investigated TRPV4 signalling by measuring [Ca²⁺]_i in HBE cells loaded with Fura–2 AM. 4α–PDD (1×10^-6M-1×10^-5M) produced a concentration-dependent increase in [Ca²⁺]_i in HBE cells (p<0.05; n=3; ANOVA + Dunnett’s test vs. vehicle control). 5’,6’-EET (1×10^-4M) also produced a significant increase in [Ca²⁺]_i in these cells (p<0.01; n=3; t test). The response to 1×10^-6M 4α-PDD in HBE cells was potentiated by pre-treatment with 1×10^-4M mouse PAR2 activating peptide (AP), but not PAR2 reverse peptide (RP; p<0.05; n=3; t test). Neither the PKC inhibitor GFX (1×10^-6M) nor the PKA inhibitor H-89 (1×10^-6M) affected this potentiation. To determine whether PAR2 sensitization affected release of the neuropeptides that mediate nociception, we measured release of calcitonin gene-related peptide (CGRP) from superfused segments of rat spinal cord. 4α-PDD (1×10^-5M and 1×10^-4M) caused a concentration-dependent increase in CGRP release, indicative of activation of nociceptive sensory neurons. The CGRP release to 1×10^-5M 4α-PDD was increased by 1×10^-5M AP, compared to 1×10^-5M RP (p<0.05; n=4; t test). Thus, PAR2 activation sensitizes the response of TRPV4 channels to the selective agonist 4α–PDD, as indicated by increased Ca²⁺ signal in HBE cells and enhanced CGRP release from spinal nociceptive neurons. Further experiments are necessary to elucidate the mechanism by which PAR2 activation produces the sensitization of TRPV4, and to determine its importance to mechanical hyperalgesia in vivo.