Bladder disorders are highly prevelant and significantly affect the quality of life. Specific treatment is not currently available due to poor understanding of the pathopfhysiology and there is an urgent need to identify novel therapeutic targets. Since the elucidation of the sensory role of the urothelium -the inner mucosal lining of the bladder, (2) there has been intense interest in identifying new physiological and pathological regulators in this structure. Oxidative stress has been shown to play a significant role in mediating many pathologies including: diabetes and cardiovascular disorders (3). Of many sources of reactive oxygen species (ROS), NADPH oxidase (Nox) is the only enzyme that produces ROS as its sole function and may serve as a promising specific therapeutic target. To date few studies have explored the role of ROS in bladder mucosa. The aim of this study was to identify the presence of Nox in the bladder and its functional significance. Guinea-pigs (male Dunkin-Hartley 450-550g) were euthanized with schedule-1 procedure in compliance with UK and EU regulations. DHE fluorescence was used to measure in situ ROS generation; cryo-sectioned samples were incubated in 2µM DHE in the presence or absence of superoxide scavenger Tiron (10mM) at 37°C (4). Immuno-fluorescence was performed on cryo-sectioned samples with Nox2 and Nox4 primary antibodies and an Alexa-586-congugated secondary antibody and examined under confocal microscope. Mucosa-intact smooth muscle strips and mucosal strips were isolated from the urinary bladders. The preparations were superfused in a HEPES-buffered Tyrode’s solution for functional measurement (5). 100µM H2O2 and 100µM Apocynin were used as exogenous ROS and Nox inhibitor. The superfusate adjacent to the tissue strip was sampled and ATP release from the tissue was measured using a luciferin-luciferase assay. DHE and immunofluorescence investigations revealed positive results for in situ ROS production and expression of Nox2 and Nox4 receptors, both in the smooth muscle and urothelium layer specifically (n=5). Application of 100µM H2O2 significantly increased ATP release from the bladder mucosa and mucosa-attached muscle respectively: (pmoles/g/min, median (25%-75% range); control: 142 (64-193) vs. 100µM H2O2: 194 (164-419); n=8, p<0.05; control: 11 (9-29) vs. 100µM H2O2: 65 (14-102); n=5; p<0.05, Wilcoxon signed rank test). ATP release was inhibited by 100µM Apocynin in mucosa-attached muscle: (pmoles/g/min, median (25%-75% range); control: 13 (7-20) vs. 100µM Apocynin: 9 (2-17); n=12, p<0.05, Wilcoxon signed rank test). These data present the first evidence for the presence of in situ ROS production and Nox2 and Nox4 in urothelium and bladder smooth muscle. Stimulation of ATP release by exogenous ROS and suppression of its release by Nox inhibitor suggest the functional relevance and the contribution from Nox enzymes.