Purpose: We established the level of adenosine triphosphate release by the urothelium in patients with painful bladder syndrome and compared it with that from the normal human bladder. Materials and Methods: Biopsies of urothelium from patients with painful bladder syndrome were subjected to stretch by 130% and 150% of the original length, and 10 Hz electric stimulation. A luciferase assay was used to quantify adenosine triphosphate release. The neurotoxin tetrodotoxin was used to block the neuronal source of adenosine triphosphate release. Results: There was a significantly greater release of adenosine triphosphate following mechanical stretch of the urothelium from painful vs control bladders. The increase in adenosine triphosphate release in painful vs control bladders was statistically significant whether expressed in absolute values (mean ± SE 3,791.4 ± 667.9 vs 77.6 ± 16.2 pM gm-1 tissue) or as an increase over baseline (282.2% ± 24.8% vs 175.4% ± 21.7%). Similarly there was a significant release of adenosine triphosphate following electrical field stimulation of the urothelium from painful vs control bladders (1,348.6 ± 278.2 vs 61.7 ± 10.1 pM gm-1 tissue, p <0.005), representing a 278% ± 41.5% vs 137.9% ± 4.4% increase above baseline (p <0.005). The source of adenosine triphosphate release was nonneuronal in 89% of painful bladders and in 84% of control bladders. Conclusions: There is a significantly increased level of adenosine triphosphate release from the urothelium of painful bladders in comparison to that from normal bladders, suggesting an important potential functional role for adenosine triphosphate in this condition.