To test the hypothesis that NPY-induced overfeeding activates compensatory responses that inhibit hypothalamic NPY gene expression, we investigated the effect of chronically administered neuropeptide Y (NPY) on plasma hormones involved in energy balance and on the level of mRNA for hypothalamic neuropeptides. After cannulation of the third cerebral ventricle, male Long-Evans rats received a 4.5-day intracerebroventricular (i.c.v.) infusion of either human NPY (12 microg per day), or synthetic cerebrospinal fluid (CSF). NPY-treated animals were either allowed ad libitum access to food or were pairfed to the intake of CSF-treated controls. In rats fed ad libitum, i.c.v. NPY induced significant increases in food intake (75%), body weight (9%), plasma insulin (150%) and plasma leptin levels (300%) as compared to the i.c.v. CSF group. Levels of plasma leptin, but not insulin, remained elevated in NPY-treated rats that were pairfed to the intake of the CSF group. NPY mRNA levels in the midregion of the arcuate nucleus (ARC) were reduced by 50% in NPY-treated rats that were allowed to overeat, but not in the pairfed group, as determined by in situ hybridization. In contrast, mRNA for corticotropin-releasing hormone (CRH) in the paraventricular nucleus (PVN) and proopiomelanocortin (POMC) in the rostral ARC were not significantly different among groups. These findings indicate that NPY-induced overfeeding suppresses ARC NPY mRNA expression, and that this effect unlikely to be mediated by a direct action of NPY, since it was abolished by limiting food intake in NPY-treated animals to that observed in controls. NPY-induced overfeeding was also associated with elevated plasma levels of leptin and insulin. The effect of these hormones to inhibit NPY gene expression may therefore have contributed to the decrease of NPY mRNA.