The present study explores the possible involvement of a purinergic mechanism in mechanosensory transduction in the bladder using P2X₃ receptor knock-out (P2X₃^−/−) and wild-type control (P2X₃^+/+) mice. Immunohistochemistry revealed abundant nerve fibers in a suburothelial plexus in the mouse bladder that are immunoreactive to anti-P2X₃. P2X₃-positive staining was completely absent in the subepithelial plexus of the P2X₃^−/− mice, whereas staining for calcitonin gene-related peptide and vanilloid receptor 1 receptors remained. Using a novel superfused mouse bladder–pelvic nerve preparation, we detected a release of ATP proportional to the extent of bladder distension in both P2X₃^+/+ and P2X₃^−/− mice, although P2X₃^−/−bladder had an increased capacity compared with that of the P2X₃^+/+ bladder. The activity of multifiber pelvic nerve afferents increased progressively during gradual bladder distension (at a rate of 0.1 ml/min). However, the bladder afferents from P2X₃^−/− mice showed an attenuated response to bladder distension. Mouse bladder afferents of P2X₃^+/+, but not P2X₃^−/−, were rapidly activated by intravesical injections of P2X agonists (ATP or α,β-methylene ATP) and subsequently showed an augmented response to bladder distension. By contrast, P2X antagonists [2′,3′-O-(2,4,6-trinitrophenyl)-ATP and pyridoxal 5-phosphate 6-azophenyl-2′,4′-disulfonic acid] and capsaicin attenuated distension-induced discharges in bladder afferents. These data strongly suggest a major sensory role for urothelially released ATP acting via P2X₃ receptors on a subpopulation of pelvic afferent fibers.