Immunofluorescence confocal microscopy demonstrated presence of Nox1, Nox2 and Nox4 subtypes in mouse bladder urothelial layer and smooth muscle, with higher intensity in the urothelium (n=6 bladders). Western blot further demonstrated specific protein bands for Nox1, Nox2 and Nox4 in bladder mucosa and smooth muscle (n=7). Lucigenin assay showed significant NADPH-dependent superoxide production in bladder tissues, sensitive to superoxide scavenger Tiron (10mM), with the main source from the mucosa. Superoxide production in bladder mucosa (RLU/mg tissue: 530±8, mean±SEM, n=16) was many fold higher than that in detrusor muscle (21±4, n=16, p<0.01), aorta (70±23, n=8, p<0.01), brain (12±3, n=7, p<0.01), kidney (95±22, n=7, p<0.01), ventricle (7±1, n=7, p<0.01) and liver (81±13, n=7, p<0.01). DHE imaging revealed positive staining in bladder tissue, with stronger intensity in the urothelium (n=8). Superoxide scavenger Tiron abolished the DHE fluorescence. The broad spectrum Nox inhibitor diphenyleneiodonium (DPI, 20μM) reduced superoxide production to 26±3% of control (n=7, p<0.01) in bladder mucosa. Mitochondria de-coupler FCCP (1μM) suppressed superoxide production to 75±11 % of control (n=7; p<0.01) in bladder mucosa. Xanthine oxidase inhibitor oxypurinol (100μM) produced no significant effect (85±13 % of control, n=8, p>0.05). Nox1 selective inhibitor NoxA1ds (5μM) inhibited superoxide production to 88±6 % of control (n=25, p<0.01). Nox2 specific inhibitor GSK2759039 (1μM) reduced superoxide production to 76±6 % of control (n=15, p<0.01). In a further set of experiments with combined inhibitors, a combination of Nox1 inhibitor NoxA1ds and Nox2 inhibitor GSK2759039 reduced the superoxide production to 71±15% of control (n=11, p<0.05), while addition of Nox1/Nox4 dual selective inhibitor GSK137831 (2μM) in the presence of the above two inhibitors reduced the superoxide production further to 53± 7% of control (p<0.01), revealing additional Nox4-selective inhibition. Application of exogenous ROS H2O2 (100µM) increased the ATP release from mucosa-attached bladder strips to 239±43% of control (n=5, p<0.01). Angiotensin II (1µM), an inflammatory mediator and Nox activator as shown in vascular tissue, increased the superoxide production (RLU/unit tissue: 275±76 to 317±112, n=16, p<0.05) and also augmented ATP release from bladder mucosa (222±20% of control, n=24, p<0.01). Angiotensin II also increased ATP release from human bladder mucosa samples (n=5).