Nociceptin or orphanin FQ (N/OFQ) and its receptor NOP1 are expressed in hypothalamic nuclei involved in energy homeostasis. N/OFQ administered by intracerebroventricular or arcuate nucleus (ARC) injection increases food intake in satiated rats. The mechanisms by which N/OFQ increases food intake are unknown. We hypothesized that N/OFQ may regulate hypothalamic neurons containing peptides involved in the control of food intake such as cocaine- and amphetamine-regulated transcript (CART), alphaMSH, neuropeptide Y (NPY), and agouti-related protein (AgRP). We investigated the ability of N/OFQ to alter the release of CART, alphaMSH, NPY, and AgRP using ex vivo medial basal hypothalamic explants. Incubation of hypothalamic explants with N/OFQ (1, 10, 100 nM) resulted in significant changes in CART and AgRP release. One hundred nanomoles N/OFQ caused a 33% decrease in release of CART (55-102) immunoreactivity (IR) and increased release of AgRP-IR to 163% but produced no change in either alphaMSH-IR or NPY-IR. Double immunocytochemistry/in situ hybridization demonstrated that CART-IR and NOP1 mRNA are colocalized throughout the hypothalamus, in particular in the paraventricular nucleus, lateral hypothalamus, zona incerta, and ARC, providing an anatomical basis for N/OFQ action on CART release. Dual in situ hybridization demonstrated that AgRP neurons in the ARC also express the NOP1 receptor. Our data suggest that nociceptin via the NOP1 receptor may increase food intake by decreasing the release of the anorectic peptide CART and increasing the release of the orexigenic peptide AgRP.