Although restraint stress accelerates colonic transit via a central corticotropin-releasing factor (CRF), the precise mechanism still remains unclear. We tested the hypothesis that restraint stress and central CRF stimulate colonic motility and transit via a vagal pathway and 5-HT3 receptors of the proximal colon in rats. 51Cr was injected via the catheter positioned in the proximal colon to measure colonic transit. The rats were subjected to a restraint stress for 90 min or received intracisternal injection of CRF. Ninety minutes after the administration of 51Cr, the entire colon was removed, and the geometric center (GC) was calculated. Four force transducers were sutured on the proximal, mid, and distal colon to record colonic motility. Restraint stress accelerated colonic transit (GC of 6.7 ± 0.4, n = 6) compared with nonrestraint controls (GC of 5.1 ± 0.2, n = 6). Intracisternal injection of CRF (1.0 μg) also accelerated colonic transit (GC of 7.0 ± 0.2, n = 6) compared with saline-injected group (GC of 4.6 ± 0.5, n = 6). Restraint stress-induced acceleration of colonic transit was reduced by perivagal capsaicin treatment. Intracisternal injection of CRF antagonists (10 μg astressin) abolished restraint stress-induced acceleration of colonic transit. Stimulated colonic transit and motility induced by restraint stress and CRF were significantly reduced by the intraluminal administration of 5-HT3 antagonist ondansetron (5 × 10−6 M; 1 ml) into the proximal colon. Restraint stress and intracisternal injection of CRF significantly increased the luminal content of 5-HT of the proximal colon. It is suggested that restraint stress stimulates colonic motility via central CRF and peripheral 5-HT3 receptors in conscious rats.