To identify regions in the hypothalamus involved in refeeding and their regulation by alpha-MSH, adult rats were subjected to a 3-d fast, and 2 h after refeeding, the distribution of c-Fos-immunoreactive neurons was elucidated. Compared with fed and fasted animals, a significant increase (P < 0.001) in the number of c-Fos-immunoreactive cells was identified in refed animals in the supraoptic nucleus, magnocellular and ventral parvocellular subdivisions of the hypothalamic paraventricular nucleus (PVNv), and the dorsal and ventral subdivisions of the dorsomedial nucleus (DMNd and DMNv, respectively). Refeeding shifted the location of c-Fos-labeled neurons from the medial to lateral arcuate where c-Fos was induced in 88.7 +/- 2.2% of alpha-MSH-containing neurons. alpha-MSH-containing axons densely innervated the PVNv, DMNd, and DMNv and organized in close apposition to the majority of refeeding-activated c-Fos-positive neurons. To test whether the melanocortin system is involved in induction of c-Fos in these regions, the melanocortin 3/4 receptor antagonist, agouti-related protein (AGRP 83-132), was administered to fasting animals just before refeeding. Compared with artificial cerebrospinal fluid, a single intracerebroventricular bolus of agouti-related protein (5 microg/5 microl) not only significantly increased the total amount of food consumed within 2 h but also nearly abolished refeeding-induced c-Fos expression in the PVNv and DMNd and partially reduced c-Fos immunoreactivity in the DMNv. We conclude that refeeding activates a subset of neurons in the PVN and DMN as a result of increased melanocortin signaling and propose that one or more of these neuronal populations mediate the potent anorexic actions of alpha-MSH.