Sheep were fed ad libitum and killed at 6 and 18 days of lactation; ad libitum-fed nonlactating sheep were killed as controls. Despite increased food intake, lactating ewes were in negative energy balance. Lactation decreased plasma leptin and adipose tissue leptin mRNA concentrations. OB-Rb gene expression, determined by in situ hybridization, was increased in the hypothalamic arcuate nucleus (ARC) and ventromedial hypothalamic nucleus (VMH) at both stages of lactation. Neuropeptide Y (NPY) was increased by lactation in both the ARC and dorsomedial hypothalamus (DMH), although increased gene expression in the DMH was only apparent at day 18 of lactation. Gene expression was decreased for cocaine-and amphetamine-regulated transcript (CART) in the ARC and VMH and for proopiomelanocortin in ARC during lactation. Agouti-related peptide gene expression was increased in the ARC, and melanocortin receptor expression was unchanged in both the ARC and VMH with lactation. Thus the hypoleptinemia of lactation may activate NPY orexigenic pathways and attenuate anorexigenic melanocortin and CART pathways in the hypothalamus to promote the hyperphagia of lactation. hypothalamic neuropeptides; appetite NUTRIENT REQUIREMENTS ARE markedly increased during lactation to meet the demands for milk production (7,70). In most mammals, these additional requirements are met primarily by increasing food intake (7,20,70), but factors regulating the hyperphagia of lactation are not well understood. In addition, in many species, the increased intake is insufficient to meet the metabolic demands, resulting in a state of negative energy balance during early lactation when body lipid reserves are mobilized (7,69). In some species such as the rat, the degree of negative energy balance is usually slight, with animals mobilizing ϳ1 g fat/day (7). Domestic ruminants, however, usually exhibit a greater degree of negative energy balance during early lactation (7, 11), which can impact on their welfare and productivity (28), yet the regulatory mechanisms have not been elucidated.It is postulated that leptin, a peptide hormone secreted by adipose tissue (75), could play a role in the hyperphagia of lactation. Leptin acts on hypothalamic neuronal systems to regulate energy balance and neuroendocrine function; in particular, a reduction in circulating leptin is a potent signal of negative energy balance, activating compensatory orexigenic pathways in the hypothalamus (3-5, 35, 64). Key targets are neurons in the hypothalamic arcuate nucleus (ARC) expressing the signaling form of the leptin receptor (OB-Rb); these are the orexigenic peptides neuropeptide Y (NPY) and agouti-related peptide (AGRP) and the anorexigenic peptides proopiomelanocortin (POMC, precursor for melanocortins) and cocaine-and amphetamine-regulated transcript (CART) (4,5,31,62,64,72). Adipose leptin gene expression and serum leptin concentrations are reported to be reduced during lactation in the monogastric rat in most (16,36,39,56,65,74), but not all (17,19,61), studies. NPY (18,21,...