Neuropeptide FF (NPFF) is a neurotransmitter known to modulate opioid-induced analgesia, sensitization, and reward. The expression of the immediate early gene c-Fos was analyzed to map the distribution of neurons whose activity is regulated by central administration of the NPFF(2)-selective agonist dNPA in naive mice and in animals who had received a systemic injection of morphine. The number of c-Fos positive nuclei was quantified in 28 brain regions. Intracerebro-ventricular injection of 1 nmol dNPA alone produced an overall inhibition of basal c-Fos expression in the brain with a statistically significant decrease in the lateral ventral part of the bed nucleus of the stria terminalis, the medial preoptic area, and the medial parvicellular part of the paraventricular nucleus of the hypothalamus. In contrast, intraperitoneal injection of morphine 5 mg.kg(-1) induced a statistically significant increase in c-Fos expression in the prelimbic cortex, the nucleus accumbens core and shell, the ventral pallidum, the lateral hypothalamus, and the nucleus of the tractus solitarius. dNPA counteracted morphine effect only in the nucleus accumbens shell and the ventral pallidum. The inhibitory effects of a low dose of dNPA in the hypothalamus and its afferents suggest that NPFF(2) receptors negatively regulate the hypothalamic-pituitary-adrenal axis in mouse. Moreover, our study identified the nucleus accumbens shell and ventral pallidum as putative sites of interaction between NPFF and opioid systems in relation with the modulation of acute morphine rewarding and locomotor effects.