Members of the muscarinic acetylcholine receptor family (M1–M5) have central roles in the regulation of many fundamental physiological functions^,. Identifying the specific receptor subtype(s) that mediate the diverse muscarinic actions of acetylcholine is of considerable therapeutic interest, but has proved difficult primarily because of a lack of subtype-selective ligands. Here we show that mice deficient in the M3 muscarinic receptor (M3R^-/- mice) display a significant decrease in food intake, reduced body weight and peripheral fat deposits, and very low levels of serum leptin and insulin. Paradoxically, hypothalamic messenger RNA levels of melanin-concentrating hormone (MCH), which are normally upregulated in fasted animals leading to an increase in food intake^,, are significantly reduced in M3R^-/- mice. Intra-cerebroventricular injection studies show that an agouti-related peptide analogue lacked orexigenic (appetite-stimulating) activity in M3R^-/- mice. However, M3R^-/- mice remained responsive to the orexigenic effects of MCH. Our data indicate that there may be a cholinergic pathway that involves M3-receptor-mediated facilitation of food intake at a site downstream of the hypothalamic leptin/melanocortin system and upstream of the MCH system.