Caffeine has marked effects on sleep, arousal and food intake. However, the neuronal mechanisms underlying these actions are not fully understood. Neuropeptide S (NPS) is a recently discovered neuropeptide regulating both sleep and feeding. Here, we examined the effect of acute and chronic caffeine treatment on the expression of neuropeptide S and its receptor (NPS-R) in the hypothalamus and brainstem of rats by using real-time PCR. Our results showed that acute caffeine treatment induces a marked decrease in the mRNA levels of NPS in the brainstem, whilst the expression levels NPS-R are increased in both hypothalamus and brainstem after caffeine treatment. The timing of both processes differs, with acute treatment affecting brainstem NPS-R expression and chronic treatment affecting hypothalamic NPS-R expression. Overall, these data suggest a possible role for the NPS system in mediating some of the behavioral effects of caffeine.