Background: Neuropeptide S (NPS) is a multifunctional regulatory factor that exhibits a potent anxiolytic activity in animal models. However, there are no reports dealing with the potential molecular relationships between the anxiolytic activity of selective serotonin reuptake inhibitors (SSRIs) and NPS signaling, especially in the context of novel stress-related neuropeptides action. The present work therefore focused on gene expression of novel stress neuropeptides in the rat brain after acute treatment with escitalopram and in combination with neuropeptide S receptor (NPSR) blockade. Methods: Studies were carried out on adult, male Sprague-Dawley rats that were divided into 5 groups: animals injected with saline (control) and experimental rats treated with escitalopram (at single dose 10mg/kg daily), escitalopram and SHA-68, a selective NPSR antagonist (at single dose 40mg/kg), SHA-68 alone and corresponding vehicle (solvent SHA-68) control. To measure anxiety-like behavior and locomotor activity the open field test (OFT) was performed. All individuals were sacrificed under anaesthesia and the whole brain excised. Total mRNA was isolated from homogenized samples of amygdala, hippocampus, hypothalamus, thalamus, cerebellum and brainstem.Real time-PCR was used for estimation of related NPS, NPSR, neuromedin U (NMU), NMU receptor 2 (NMUR2) and nesfatin-1 precursor nucleobindin-2 (NUCB2) gene expression.Results: Acute escitalopram administration affects the local expression of the examined neuropeptides mRNA in a varied manner depending on brain location. An increase of NPSR and NUCB2 mRNA expression in the hypothalamus and brainstem was abolished by SHA-68 coadministration, while NMU mRNA expression was upregulated after NPSR blockade in the hippocampus and cerebellum. Conclusions: Pharmacological effects of escitalopram may be connected with local NPSR-related alterations in NPS/NMU/NMUR2 and nesfatin-1 gene expression at the level of selected rat brain regions. A novel alternative mode of SSRI action can be therefore cautiously proposed.