The mechanosensitive Ca2+ permeable cation channel TRPV4 is expressed in the urothelium and detrusor smooth muscle (DSM) of rodent bladders (Thorneloe et al., 2008). Urothelial stretch during bladder filling activates TRPV4 and produces a Ca2+-dependent release of ATP (Mochizuki et al., 2009). This ATP release activates P2X3 receptors on sensory nerves to produce a sensation of bladder filling (Vlaskovska et al., 2001). Activation of TRPV4 in the DSM by the selective agonist GSK1016790A (10-100nM) produces a slow and sustained contraction, which can be inhibited by nifedipine (Thorneloe et al., 2008). We hypothesised that deletion of the TRPV4 channel would reduce the contractility of isolated DSM strips by reducing the intracellular [Ca2+]. Male C57BL/6 wild type (+/+) and TRPV4 knockout (-/-) mice (22-30g) were used in this study. Bladders were collected, sectioned above the level of the ureters, then cut into 2 longitudinal strips. These were suspended under 0.5g tension in a 10ml organ bath containing Krebs’ solution (pH7.4) at 37oC and bubbled with a mixture of 95% O2 and 5% CO2. Changes in isometric force were recorded using a Power Lab v.4 system (AD Instruments, UK). Concentration-response curves to the muscarinic agonist carbachol (cumulative) and the P2X receptor agonist α,β-methylene ATP (non-cumulative) were constructed. Following pre-contraction of the tissue strips with KCl, a concentration-response curve was obtained for relaxation to the β-adrenergic agonist isoprenaline. Frequency-response curves to electrical field stimulation (EFS: 10s; 80V; pulse width 1ms) were also obtained. Emax and pEC50 values (stated as mean ± S.E.M) were compared by unpaired t-tests. Maximal contractions to carbachol were significantly increased in TRPV4 -/- strips (1nM-30μM; +/+ = 0.029±0.004 vs -/- = 0.077±0.011g/mg, p<0.05, n=5) with no effect on pEC50 values. Contractions to EFS (4,8,16,32Hz) were also increased in TRPV4 -/- strips compared to +/+ strips. Contractions to α,β-methylene ATP (1-10μM, n=4) were unaltered by loss of TRPV4. Maximal relaxation to isoprenaline was significantly decreased in TRPV4 -/- strips (1nM-30µM; +/+ = 61.6±8.3 vs -/- = 41.8±3.8 % of KCl contraction, p<0.05, n=5) with no effect on pEC50 values. Pretreatment with the P2 receptor antagonist suramin (100µM) or the NOS inhibitor L-NAME (100µM) had no effect on the contractions to carbachol in bladder strips from wild type mice. These data suggest that muscarinic and β-adrenergic receptors activate a TRPV4-dependent mechanism, probably via urothelial TRPV4, that inhibits DSM contraction. This inhibitory mechanism is not P2 receptor or NO dependent. Activation of P2X receptors causes DSM contraction without modulation by TRPV4.