Overactive bladder syndrome and interstitial cystitis are age-related disorders affecting millions worldwide but the factors that predispose aging bladders to these chronic bladder diseases are poorly understood. The role of oxidative stress and reactive oxygen species (ROS) have been postulated to an underlying mechanism for aging and many age-associated disorders. NADPH oxidases (Nox enzymes) are professional ROS-generating enzymes in the body that produce superoxide as their sole function and thus serve as specific ROS-controlling therapeutic target with little side effect on physiological oxidation. Nox-derived ROS have been shown to contribute to many age-related chronic conditions, but little is known about its role in the bladder, in particular in the inner-lining epithelial layer – the urothelium. Urothelium has been recognised as a new sensory structure and its dysfunction may hold key to understanding age-related bladder disorders. The purpose of this work was to determine Nox-dependent superoxide generation in bladder tissue in response to bladder pathology-related inflammatory mediators. Wild-type C57BL/6 J mice were maintained under standard feeding conditions and euthanized in accordance with UK and EU regulations. Bladders were isolated, bladder mucosa and smooth muscle layer were dissected under microscopic guidance. Nox proteins were determined by Western blot with Nox subtype-specific antibodies. NADPH-dependent superoxide production was measured by lucigenin-enhanced luminance. ATP release, a sensory transduction process of the urothelium, was quantified by luciferin-luciferase assay. Data are expressed as mean±SEM. Two group means were analysed by t-test (paired or non-paired). Multiple means were tested by ANOVA and pairwise comparisons. Major Nox subtypes (Nox 2, Nox1 and Nox4) were shown to be expressed within the bladder, specifically within the bladder mucosa, a structure containing the urothelium and the lamina propria (n=5-8 bladders). The superoxide generation was significantly higher in bladder mucosa than in smooth muscle layer (relative light unit [RLU]/mg tissue: 201±23 vs 37±4 for mucosa and smooth muscle respectively, n=45, p<0.01). The bladder inflammation-associated mediator bradykinin (10µM) increased the superoxide production in bladder mucosa to 250±34% of control (n=11, p<0.05). Bladder obstruction and hypoxia-related inflammatory mediator endothelin-1 enhanced the superoxide production to 231±43% of control (n=12, p<0.05). Further experiments showed that these inflammatory mediators also augmented ATP release from the urothelium. These results demonstrate a potential role for Nox-derived ROS in chronic inflammation in response to inflammatory mediators encountered in bladder pathologies and suggest pathological significance. The effect of these mediators on urothelial ATP release supports functional relevance.