Non steroidal anti-inflammatory, cyclooxygenase (COX) inhibitors that function to reduce prostaglandin E2 (PGE2) production, have been widely reported as effective agents in models of urinary bladder overactivity. We therefore investigated a potential role for the PGE2 receptor, EP3, in urinary bladder function by performing conscious, freely moving cystometry on EP3 receptor knock-out (KO) mice. EP3 KO mice demonstrated an enhanced bladder capacity compared to wild type (WT) mice (~185% of WT) under control conditions, based on larger voided and infused bladder volumes. Infusion of the EP3 receptor agonist GR63799X into the bladder of WT mice reduced the bladder capacity. This was ineffective in EP3 KO mice that demonstrated a time-dependent increase in bladder capacity with GR63799X, an effect similar to that observed with vehicle in both genotypes. In addition, infusion of PGE2 into WT mice induced bladder overactivity, an effect that was significantly blunted in the EP3 KO mice. The data reported here provide the first evidence supporting a functional role for EP3 receptors in normal urinary bladder function, and implicate EP3 as a contributor to bladder overactivity during pathological conditions of enhanced PGE2 production, as reported previously in overactive bladder patients.