Neuropeptide S (NPS) is the endogenous ligand of a previously orphan receptor now named NPSR. In the brain NPS regulates several biological functions including anxiety, arousal, locomotion, food intake, learning and memory, pain and drug abuse. Mice lacking the NPSR gene (NPSR(−/−)) represent an useful tool to investigate the neurobiology of the NPS/NPSR system. NPSR(−/−) mice have been generated in a 129S6/SvEv genetic background. In the present study we generated CD-1 congenic NPSR(+/+) and NPSR(−/−) mice and investigated their phenotype and sensitivity to NPS in various behavioural assays. The phenotype analysis revealed no locomotor differences between NPSR(+/+) and NPSR(−/−) mice. The behaviour of NPSR(+/+) and NPSR(−/−) mice in the righting reflex test was superimposable. No differences were recorded between the two genotypes in the elevated plus maze, open field and stress-induced hyperthermia tests, with the exception of rearing behaviour that was reduced in knockout animals. Moreover the behaviour of NPSR(+/+) and NPSR(−/−) mice in the forced swimming, novel object recognition and formalin assays was similar. The stimulatory effects of NPS in the locomotor activity test and its anxiolytic-like actions in the elevated plus maze and open field assays were evident in NPSR(+/+) but not NPSR(−/−) animals. In conclusion, the present study indicates that the NPS/NPSR system does not tonically control locomotion, sensitivity to diazepam, anxiety, depressive-like behaviours, memory and pain transmission in mice. Furthermore our results clearly show that the product of the NPSR gene represents the mandatory protein for all the NPS biological effects so far described.