The neurotransmitter neuropeptide Y (NPY) is abundant in the hypothalamus where its actions include the potent stimulation of food intake. The peripheral metabolic and hormonal signals involved in its regulation are not clear. The aim of this study was to investigate possible actions of corticosteroids and insulin on hypothalamic NPY synthesis and content in vivo. We measured NPY content in individual hypothalamic nuclei, and hypothalamic NPY mRNA by Northern blotting in whole hypothalamus in rats treated with dexamethasone (0.4 mg/kg/day) and dexamethasone plus insulin (60 U/kg/day), compared to controls. The effect of stopping dexamethasone treatment was also studied. Dexamethasone treatment produced significant increases in NPY in the paraventricular (11.0 ± 1.3 vs. 7.1 ± 0.4 fmol/µg protein, p < 0.05) and arcuate (6.2 ± 0.3 vs. 3.8 ± 0.2 fmol/µg protein, p < 0.001)nuclei of the hypothalamus, paralleled by a 38% increase in total hypothalamic NPY mRNA (p < 0.05). These changes were not seen in the group treated with dexamethasone plus insulin. In the group in whom dexamethasone was stopped, NPY mRNA was unchanged compared to controls, but peptide content remained increased in the arcuate but not the paraventricular nucleus (arcuate 7.7 ± 0.7 vs. 5.5 ± 0.7, PVN 4.9 ± 1.0 vs. 4.7 ± 0.9 fmol/µg protein). Thus hypothalamic NPY and its mRNA were increased by cortico-steroid administration, and this effect was prevented by systemic insulin treatment. This dual regulatory system for hypothalamic NPY may be important in the control of food intake by corticosteroids and insulin.