Rationale: Overactive Bladder (OAB) is an idiopathic condition, characterized by urgency, urinary frequency and urgency incontinence, in the absence of routinely traceable urinary infection. We have described microscopic pyuria (≥10 wbc μl-1) in patients suffering from the worst symptoms. It is established that inflammation is associated with increased ATP release from epithelial cells, and extracellular ATP originating from the urothelium following increased hydrostatic pressure, is a mediator of bladder sensation. Objectives: Here, using bladder-biopsy samples, we have investigated urothelial ATP signaling in OAB patients with microscopic pyuria. Findings: Basal, but not stretch-evoked, release of ATP was significantly greater from urothelium of OAB patients with pyuria than from non-OAB patients or OAB patients without pyuria (<10 wbc μl-1). Basal ATP release from urothelium of OAB patients with pyuria was inhibited by the P2 receptor antagonist suramin and abolished by the hemichannel blocker carbenoxolone, which differed from stretch-activated ATP release. Altered P2 receptor expression was evident in urothelium from pyuric OAB patients. Furthermore, intracellular bacteria were visualized in shed urothelial cells from ~80% of OAB patients with pyuria. Conclusions: These data suggest that increased ATP release from the urothelium, involving bacterial colonization, may play a role in the heightened symptoms associated with pyuric OAB patients.