The urothelium is a newly-recognised sensory structure that releases ATP to excite the afferents. Evidence suggests that transient receptor potential vallinoid 4 (TRPV4) channels present in the urothelium may serve as key sensory molecules, responding to noxious stimuli. The mode of action of these channels in the urothelium is poorly understood, particularly in native tissue. This study aimed to determine the role of TRPV4 in ATP release and the underlying mechanisms in intact urothelium. Urothelial and urothelium-attached smooth muscle strips (full thickness) were isolated from guinea-pig bladders (male Dunkin-Hartley, schedule-1) and superfused with a Tyrode’s solution. The superfusate adjacent to tissue was sampled and measured for ATP release using a luciferin-luciferase assay. TRPV4 activator GSK1016790A (GSK, 1μM) triggered significant increase in ATP release in both tissues (urothelium: 246±24% of control; full thick strip: 191±16% of control; mean ± SEM, n=8, p<0.05, Wilcoxon’s). This effect was inhibited by TRPV4 antagonist HC-067047 (HC)(urothelium: a reduction from 232±25% to 102±6%; full thick strip: 175±13% to 113±7%, n=7, p<0.05), demonstrating specific activation of TRPV4 channels. GSK-triggered ATP release was largely abolished in a Ca2+ free Tyrode’s solution (urothelium: reduced from 253±35% to 113±5%; full thick strip: 190±14% to 120±12%, n=7, p<0.05). Hemichannel blocker carbenoxolone (100µΜ) caused significant reduction of GSK-triggered ATP release in both tissues (urothelium: a decrease from 317±49% to 146±25 %, full thick strip: 329±48 % to 156±44 %, n=9, p<0.05). In contrast, vesicular transport inhibitor brefeldin A (10μM) and chloride channel blocker 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB, 100μM) had no significant effect (brefeldin A; urothelium: GSK 297±66% vs. GSK/brefeldin A 249±18%; full thick strip: GSK 227±28% vs. GSK/brefeldin A 227±25%, n=5, p>0.05; NPPB; urothelium: GSK 194±24% vs. GSK/NPPB 276±35%; full thick strip: GSK 240±21% vs. GSK/NPPB 230±39 %, n=6, p>0.05). Application of GSK1016790A directly to denuded smooth muscle generated only a moderate increase in ATP release (140±25% control, n=12, p<0.05), suggesting that the ATP release triggered by TRPV4 activation originates almost exclusively from the urothelium. Finally application of HC to un-stimulated full-thickness preparations reduced basal ATP release (77±10% of control, p<0.05), suggesting intrinsic activation of TRPV4 channels in the tissue. These results provide the first evidence that selective TRPV4 activation generates significant ATP release from the urothelium in native bladder tissue; this effect is mediated by extracellular Ca2+ entry and connexin conductive pathways with little contribution from vesicular transport and chloride channels. Intrinsic TRPV4 activity and associated ATP release demonstrate further physiological relevance of TRPV4 in bladder function.