The excitatory roles of EP3 receptors at the peripheral afferent nerve innervating the rat urinary bladder have been evaluated by using the selective EP3 antagonist, (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). The bladder rhythmic contraction model and a bladder pain model measuring the visceromotor reflex (VMR) to urinary bladder distension (UBD) have been used to evaluate DG041 in female rats. In addition, male rats (SHR, WKY and SD) were anesthetized with sodium pentobarbital and primary afferent fibers in the L6 dorsal root were isolated for recording the inhibitory response to UBD following IV injection of DG041. IV injection of DG041 (10 mg/kg), a peripherally restricted EP3 receptor antagonist, significantly reduced the frequency of bladder rhythmic contraction and inhibited the VMR response to bladder distension. The magnitude of reduction of the VMR response was not different in the different strains of rats (SD, SHR, and WKY). Furthermore, quantitative characterization of the mechanosensitive properties of bladder afferent nerves in SHR, WKY and SD rats did not show the SHR to be supersensitive to bladder distension. DG041 selectively attenuated responses of mechanosensitive afferent nerves to UBD, with strong suppression on the slow conducting, high threshold afferent fibers, with equivalent activity in the three strains. We conclude that sensitization of afferent nerve activity was not one of the mechanisms of bladder hypersensitivity in SHR. EP3 receptors are involved in the regulation of bladder micturition and bladder nociception at the peripheral level.