We investigated the presence of EP₁ receptor in the urothelium and its role in micturition reflex by examining the effect of intravesical administration of prostaglandin E₂ (PGE₂), an EP₁ agonist (ONO-DI-004), acetic acid, and capsaicin. Age-matched EP₁-KO mice and C57BL/6 wild-type (WT) mice were used. Western blots and standard immunohistochemical procedures were performed. Cystometrygram (CMG) was performed without anesthesia in a restraining cage. ATP release from the cultured urothelium cells was performed using luciferin-luciferase luminometry. The EP₁ receptor was found to be present in the urothelium. In WT mice, PGE₂ infusion shortened the intercontraction interval (ICI) in a dose-dependent fashion; however, it did not alter the ICI in EP₁-KO mice. The EP₁ agonist significantly shortened the ICI in WT mice, but not in EP₁-KO mice. Acetic acid and capsaicin shortened the ICI in both WT mice and EP₁-KO mice. EP₁ agonist, PGE₂ and capsaicin provoked ATP release from cultured urothelial cells. These results suggest that EP₁ receptor was present in bladder urothelium, and could be activated by PGE₂ to release ATP. EP₁ receptor in urothelium might be important for reflex voiding in pathological conditions.