Brännstrom’s hydrodynamic theory postulated that dental pain results from the activation of dental sensory neurons in response to dentinal fluid movement under thermal expansion and contraction. It is not clear whether this is a direct mechanotransduction by neurons or whether other dental structures contribute to the mechanosensation. Immunohistochemical and functional studies suggest the expression of thermosensitive and mechanosensitive Transient Receptor Potential (TRP) A1, M8 and V1 Ca2+ permeable cation channels in human and rat odontoblasts. However, a mechanism by which odontoblasts could modulate neuronal activity has not been demonstrated. In this study we investigated functional TRP channel expression in human odontoblast-like cells (OBs) and aimed to identify chemical transmitter release in response to TRP channel activity. Human immortalized dental pulp cells were driven towards an odontoblast-like phenotype by culture in media conditioned with ascorbic acid, β-glycerophosphate and dexamethasone (Egbuniwe et al., 2011). Functional expression of TRP channels was determined using RT-PCR and ratiometric calcium imaging with Fura-2. ATP release was measured using a luciferin-luciferase assay. Expression of mRNA for TRPA1, TRPV1 and TRPV4 but not TRPM8 was detected in OBs by RT-PCR. Exposure to the TRPA1 agonists allyl isothiocyanate (AITC) and cinnamaldehyde (1µM-500µM) or the TRPV4 agonist GSK1016790A (1nM-1µM) caused a concentration-dependent increase in intracellular Ca2+ concentration that was inhibited by the selective antagonists HC030031 (TRPA1) and HC067047 (TRPV4) (n=5-7 independent triplicate measurements). In contrast, exposure to the TRPV1 agonist capsaicin (1nM-1µM) or the TRPM8 agonist icilin (1nM-1µM) had no effect on intracellular Ca2+ concentration (n=3). Treatment for 15 min with AITC (500µM; 28.2±5.1 vs. 10.4±4.5 nM), cinnamaldehyde (500µM; 29.9±8.6 vs. 9.0±2.3 nM) or GSK1016790A (1µM; 15.9±3.5 vs. 6.5±1.2 nM) caused an increase in ATP concentration in culture medium, compared to vehicle treatments (mean±S.E.M; p<0.05; t-test with Welch’s correction; n=9-10). These data demonstrate that activation of the mechanosensitive TRPA1 and TRPV4 channels in human odontoblast-like cells can stimulate ATP release, which may modulate the activity of adjacent dental sensory neurons in situ via purinergic receptors. We were unable to confirm the presence of thermosensitive TRPV1 and TRPM8 that has previously been reported in odontoblasts.