Neuropeptide Y (NPY) and NPY mRNA are increased in the hypothalamic arcuate nucleus of rats after prolonged fasting but it is not known if NPY mRNA is also affected by more moderate fasting. NPY mRNA was quantified by in situ hybridization at five levels through the rostral/caudal axis of the arcuate nucleus and was found to be expressed in a markedly heterogenous fashion. NPY mRNA increased significantly in the mid portion of the arcuate nucleus after 24 h of fasting and further increased with fasting for 48 and 96 h. At the most caudal level of the arcuate nucleus, as well as in the dorsomedial nucleus of the hypothalamus and the reticular nucleus of the thalamus, NPY mRNA was unaffected by fasting. Serum insulin decreased after 24 h of fasting and decreased further with more prolonged fasting. Insulin receptor binding, which was highest in the same region of the arcuate nucleus that expressed NPY mRNA, was unaffected by fasting. These data show that NPY mRNA levels in the arcuate nucleus are sensitive to a relatively minor degree of food deprivation and are consistent with the hypothesis that circulating insulin modulates NPY mRNA expression in the arcuate nucleus during mild to prolonged fasting.