The effect of a novel peptide, orexin A, on longitudinal muscle of ICR mouse small intestine was examined in vitro. Exogenous orexin A induced a transient contraction in duodenal, jejunal and ileal segments. Atropine and tetrodotoxin completely inhibited the contractions. Contraction of longitudinal muscle of jejunal segments induced by electrical field stimulation was still observed after the jejunal segment had been desensitized to orexin A, suggesting that orexin A is not a final neurotransmitter to induce the contraction. On the other hand, in the presence of atropine and guanethidine, orexin A induced a transient gradual relaxation in duodenal, jejunal and ileal segments. Electrical field stimulation also induced significant relaxation of the muscle in jejunal segments. The electrical field stimulation-induced relaxation was inhibited by 55% after the desensitization of the segments to orexin A. Although the electrical field stimulation-induced relaxation was inhibited by 47% by a nitric oxide synthesis inhibitor, NG-nitro-L-arginine (L-NOARG), orexin desensitization did not affect the relaxation which persisted after L-NOARG treatment. The exogenous orexin A-induced relaxation was completely inhibited by L-NOARG. The results suggest that orexin A partially mediates nonadrenergic, noncholinergic (NANC) relaxation via activation of nitrergic neurones in longitudinal muscle of ICR mouse small intestine.