Neuropeptide S (NPS) is the endogenous ligand for the previously orphan G-protein-coupled-receptor, now termed NPS receptor (NPSR). NPS has both anxiolytic and pro-arousal properties and decreases food intake. In this work we use a rat model of palatable food intake to test in vivo different analogs of human NPS developed in our laboratories and characterized in previous in vitro experiments as partial agonists ([Ala(3)]NPS and [Aib(5)]NPS), or antagonists ([D-Cys((t)Bu)(5)]NPS and [(t)Bu-D-Gly(5)]NPS). Our results confirmed that intracerebroventricular (ICV) injection of NPS (1 nmol) decreases standard chow intake in food restricted rats as well as in freely feeding animals fed with standard or palatable food diets. [Aib(5)]NPS (30 and 60 nmol), like NPS, reduced palatable food intake, thus confirming in vivo its ability to activate NPSR. [Ala(3)]NPS (60 nmol) did not affect palatable food intake per se but blocked the anorectic effect of NPS, thus suggesting its ability to function as an antagonist in this model. Finally, [D-Cys((t)Bu)(5)]NPS (20-60 nmol) and [(t)Bu-D-Gly(5)]NPS (10-30 nmol), described in previous in vitro studies as pure NPSR antagonists, did not affect palatable food intake when given alone, but fully blocked the anorectic effect of NPS. These results provide an important characterization of the pharmacological properties of these NPS analogs in vivo. Of particular relevance are the data showing that [D-Cys((t)Bu)(5)]NPS and [(t)Bu-D-Gly(5)]NPS behave as pure antagonists at NPSR regulating food intake, indicating that these molecules are suitable tools for further investigation of the physiopharmacology of the NPS/NPSR system.