Food deprivation elevates both neuropeptide Y (NPY) gene expression in the basomedial hypothalamus and NPY levels in the paraventricular nucleus (PVN). To gain a better understanding of the dietary control of NPY, we systematically examined the effects of macronutrient restriction on the gene expression of NPY in the basomedial hypothalamus and NPY content in the PVN. Rats were fed one of eight different diets for 12 d. The control group was fed a modified AIN-76 diet. One group was fed the modified AIN-76 diet but was restricted in energy by 50%. Six isocaloric (to the control diet) test diets were prepared, each with an individual macronutrient reduced by 50%. Neuropeptide Y gene expression was elevated in protein-restricted animals as well as in energy-restricted animals. Carbohydrate or fat restriction seemed to have no effect on NPY gene expression. Though the NPY content in the PVN was not different between any groups, two findings were consistent with greater NPY release in protein-restricted animals. Animals fed a low protein diet were hyperphagic and had a greater amount of body fat. This study suggests that low levels of dietary protein may have a role in the regulation of NPY gene expression.