Neuropeptide Y (NPY) is known to stimulate eating behavior and to be related to behavioral patterns of carbohydrate ingestion. The present report investigates this relationship further to: (1) characterize the specific NPY projection activated in different dietary paradigms; (2) understand associated changes in circulating hormones that may mediate dietary effects on NPY neurons; and (3) determine whether endogenous NPY in conditions with macronutrient diets can be linked to body fat. Male albino Sprague-Dawley rats were tested in two feeding paradigms, one in which the rats were given a choice of the macronutrients, carbohydrate, fat or protein, or the other involving a single diet varying in carbohydrate of fat content. These studies consistently demonstrated a close association between the ingestion of carbohydrate and NPY levels, specifically in the arcuate nucleus (ARC) and medial portion of the paraventricular nucleus (PVN) of the hypothalamus. In addition to revealing increased NPY activity in animals that naturally select high carbohydrate when given a choice of macronutrients, a single diet with 65% carbohydrate (10% fat), compared to a control diet with 45% carbohydrate (30% fat), significantly potentiates NPY gene expression and NPY-immunoreactivity, as determined by in situ hybridization and immunohistochemistry. A further lowering of carbohydrate to 15% has little effect on NPY. Studies of medial hypothalamic fragments in vitro also reveal enhanced NPY release from hypothalamic tissue taken from rats maintained on high-carbohydrate diet. Together with NPY, circulating corticosterone (CORT) levels are also highest in a high-carbohydrate condition and positively correlated with NPY in the ARC. An association between NPY and adiposity in these dietary conditions is indicated by significantly higher levels of NPY in the medial PVN in rats with high body fat, whether consuming a high-carbohydrate of high-fat diet. This evidence, linking NPY to carbohydrate intake and circulating CORT, suggests a role for this peptide in glucose homeostasis that is normally exhibited under conditions when carbohydrate stores are low. Disturbances in this homeostatic process, associated with hyperinsulinemia and higher levels of NPY, become evident with only a moderate rise in body fat on a high-carbohydrate as well as high-fat diet.