Background: There is increased evidence to suggest a role for nonadrenergic-noncholinergic neurotransmission in the pathogenesis of bladder dysfunction. Objective: In this set of experiments, we have assessed the contribution of the urothelium to purinergic activity by quantifying the amount of adenosine triphosphate (ATP) released from the urothelium of patients with idiopathic detrusor overactivity (IDO) and with neurogenic detrusor overactivity (NDO) and comparing these releases to those of controls. Design, setting, and participants: Bladder tissue with urodynamically and clinically proven NDO (n = 8) and IDO (n = 8) were included in this study. The carefully dissected urothelium was stimulated by mechanically stretching as well as electrically stimulating and the ATP; thus, release was quantified. Measurements: We used a Lucy Anthos 1 luminometre (Anthos Labtec Instruments GmBH, Wals, Austria) to perform the assay. The results were analysed using Stingray software (Dazdaq Ltd, Brighton, UK). Results and limitations: Both mechanical stretch and electric field stimulation (EFS) led to increased ATP release in both sets of tissues with overactivity compared to the controls; this rise was even more significant for the IDO urothelium (2416.7 ± 479.8 pmol/g [p < 0.005]) than for the NDO urothelium (133.1 ± 22.4 pmol/g [p < 0.01]); values for the controls were 77.6 ± 16.2 pmol/g. ATP release following mechanical stretch was more sensitive to tetrodotoxin in bladders with NDO compared to those with IDO as well as to the controls, with ATP levels falling from 233.5 ± 20.7 pmol/g to 107.2 ± 11.6 pmol/g, expressed as percentage of basal levels (p < 0.002). The experiments were performed in vitro, and the female patients were a mix of peri- and postmenopausal states. Conclusions: These experiments suggested a significant rise in ATP release from the urothelium of bladders with NDO as well as those with IDO in comparison to controls. Most of the ATP released from bladders with NDO is primarily from neuronal sources.