The involvement of orexin in neural pathways for peristalsis was examined in mouse jejunal segments. Localized distension of the segments using a small balloon resulted in ascending contraction and descending relaxation. Ascending contraction was abolished by atropine and tetrodotoxin. Desensitization to orexin A (OXA) and SB-334867-A, an orexin-1 receptor antagonist, significantly inhibited ascending contraction. Hexamethonium also produced a significant inhibition. Exogenous administration of either OXA or nicotine induced a transient contraction that was completely inhibited by atropine and tetrodotoxin. The OXA-induced contraction was significantly inhibited by hexamethonium and SB-334867-A, whereas the nicotine-induced contraction was not inhibited by SB-334867-A. Descending relaxation was either partially or completely inhibited by l-nitroarginine and tetrodotoxin, respectively. Both SB-334867-A and hexamethonium partially inhibited descending relaxation. A combination of SB-334867-A and hexamethonium had an additive inhibitory effect on descending relaxation. Exogenous OXA, in the presence of atropine, induced a relaxation that was significantly inhibited by both l-nitroarginine and SB-334867-A, but not by hexamethonium. Nicotine in the presence of atropine relaxed the jejunal segment. SB-334867-A, unlike hexamethonium, did not affect nicotine-induced relaxation. These results suggest that OXA plays an important role in the ascending and descending neural reflexes in the mouse jejunum.