Prostaglandin EP3 receptors in the central nervous system (CNS), may exert an excitatory effect on urinary bladder function via modulation of bladder afferent pathways. We have studied this action, using two EP3 antagonists, (2E)-3-{1-[(2,4-dichlorophenyl)methyl]-5-fluoro-3-methyl-1H-indol-7-yl}-N-[(4,5-dichloro-2-thienyl)sulfonyl]-2-propenamide (DG041) and (2E)-N-{[5-bromo-2-(methyloxy)phenyl]sulfonyl}-3-[2-(2-naphthalenylmethyl)phenyl]-2-propenamide (CM9). DG041 and CM9 were proven to be selective EP3 antagonists, using radioligand binding and functional fluorescent imaging plate reader (FLIPR) assays. Their effects on volume-induced rhythmic bladder contraction and the visceromotor reflex (VMR) response to urinary bladder distension (UBD) have been evaluated in female rats following intrathecal (i.t.) or intracerebroventricular (i.c.v.) administration. Both DG041 and CM9 showed a high affinity for the EP3 receptors at the subnanomolar concentrations without significant selectivity for any splice variants. At the human EP3C receptor, both inhibited calcium influx produced by the non-selective agonist PGE2. Following i.t. or i.c.v. administration both CM9 and DG041 dose-dependently reduced the frequency, but not the amplitude of the bladder rhythmic contraction. With i.t. administration, DG041 and CM9 produced a long lasting and robust inhibition on the VMR response to UBD, whereas with i.c.v. injections, both compounds elicited only a transient reduction of the VMR response to bladder distension. These data support that EP3 receptors are involved in bladder micturition at supraspinal and spinal centers and in bladder nociception at the spinal cord. A centrally acting EP3 receptor antagonist may be useful in the control of detrusor overactivity and/or pain associated with bladder disorders.