Visceral hypersensitivity and stress have been implicated in the pathophysiology of functional gastrointestinal disorders. We used a selective vasopressin 3 (V₃) receptor antagonist, SSR149415, to investigate the involvement of the vasopressin (AVP)/V₃ signaling system in the development of stress-induced visceral hyperalgesia in rats. Rats were exposed to a daily 1-h session of water avoidance stress (WAS) or sham WAS for 10 consecutive days. The visceromotor response to phasic colorectal distension (CRD, 10-60 mmHg) was assessed before and after stress. Animals were treated daily with SSR149415 (0.3, 1 or 3 mg/kg, ip, 30 min before each WAS/sham WAS session) or with a single dose of SSR149415 (1 mg/kg, ip) or the selective CRF₁ antagonist, DMP-696, (30 mg/kg, po) before CRD at day 11. Effects of a single dose of SSR149415 (10 mg/kg, iv) on acute mechanical sensitization during repetitive CRD (12 distensions at 80 mmHg) were also assessed. In vehicle-treated rats, repeated WAS increased the response to CRD, indicating visceral hypersensitivity. Repeated administration of SSR149415 at 1 or 3 mg/kg completely prevented stress-induced visceral hyperalgesia. Similarly, a single dose of DMP-696 or SSR149415 completely blocked hyperalgesic responses during CRD. In contrast, a single dose of SSR149415 did not affect the acute hyperalgesic responses induced by repeated, noxious distension. These data support a major role for V₃ receptors in repeated psychological stress-induced visceral hyperalgesia and suggest that pharmacological manipulation of the AVP/V₃ pathway might represent an attractive alternative to the CRF/CRF₁ pathway for the treatment of chronic stress-related gastrointestinal disorders.