Neuropeptide S (NPS) and its receptor are expressed by several cell types within the gastrointestinal (GI) tract, such as the epithelium, smooth muscle cells, submucosal neurons and enteroendocrine cells, in both rat and man. Polymorphisms of the NPS receptor are linked to increased risk of inflammatory bowel disease (IBD) as well as motor and sensory disturbances of the gut, suggesting a role for NPS in GI disorders. The aim of the present study was to elucidate the actions of NPS on duodenal mucosal barrier function and motility. Male Sprague-Dawley (SD) rats (n=36; 300-350 g) were anaesthetized with thiobutabarbital 120 mg/kg body weight intraperitoneally The surgical and experimental protocol and methodology used have been described previously [1]. A brief summary is given here. A ~30 mm segment of the proximal duodenum was perfused in situ. The effects of intravenous (IV) administration of NPS and NG-nitro-L-arginine methyl ester (L-NAME) were investigated on mucosal paracellular permeability, bicarbonate secretion (DBS), motility and net fluid flux. In addition, studies of migrating motor complexes were carried out in SD rats (n=30; 300-350 g) with electrodes implanted in the small bowel. After baseline recordings with saline infusion, NPS was infused IV for 60 min and effects on myoelectric activity recorded. To relate the findings in rat to the human GI tract, circular muscle strips of human small intestine (n=7) was studied in an organ bath. The human tissue specimens were excised from patients during surgery for stomach or intestinal resection. All patients gave informed consent to enter the study. Tension changes were measured after stimulation with 1.0-20 nM NPS with and without electric field stimulation (EFS). Values are mean ± SEM, compared by 1-way ANOVA or paired t test when appropriate. NPS (0.5, 5.0 and 50 nmol kg-1 h-1) caused dose-dependent decrease in duodenal motility and paracellular permeability (e.g. from 431 ± 66 to 145 ± 51 AUC 10 min-1 and from 0.21 ± 0.03 to 0.09 ± 0.02 ml min-1100g-1, n=10, P<0.001 respectively). NPS did not induce any changes in DBS or net-fluid-flux. Pre-treating animals with L-NAME (3.0 mg kg-1 bolus dose followed by infusion of 1.0 mg kg-1 h-1) abolished the effect of NPS on duodenal motility, but did not change that on permeability. Similarly, NPS at 1.0 nmol kg-1min-1 increased irregular spiking, while 4.0 nmol kg-1min-1 (each dose n=6) significantly reduced spiking activity and increased the MMC cycle length (P=0.005). In human smooth muscle strips, NPS (5.0 nM, n=7) dampened small intestine circular muscle contractility with and without EFS. In colonic muscle strips (NPS 5.0 nM) only sporadic dampening without EFS, but more consistent dampening (NPS 1.0 nM, n=5 P=0.04) with EFS, was observed. In conclusion, these findings reveal evidence that NPS significantly decreases small intestinal motility and contractility in tissue of both human and rat origin. In addition, NPS decreases paracellular permeability. L-NAME abolished the effect of NPS on motility but was without effect on that to permeability. These results suggest important roles of NPS in the regulation of GI barrier functions and motility.