Lactation is characterized by extreme hyperphagia and negative energy balance resulting from a large energy drain due to milk production and by a suppression of cyclic ovarian function. Increases in neuropeptide Y (NPY) and agouti-related protein and a decrease in proopiomelanocortin expression in the arcuate nucleus of hypothalamus (ARH) may contribute to the hyperphagia to maintain energy balance and to the suppression of LH secretion associated with lactation. However, little is known about the full extent of neuroendocrine changes in the ARH that may contribute to the various adaptations occurring during lactation. To address this issue, we used Affymetrix microarray to acquire a reliable profile of the lactation-induced transcriptional changes in micropunches containing the ARH and a portion of the ventromedial nucleus of the hypothalamus (VMH). Using high stringency criteria, 12 genes were identified as being differentially regulated during lactation and an additional 10 genes and 3 transcribed sequences were identified using moderate stringency criteria. Changes in NPY, enkephalin, tyrosine hydroxylase and dynorphin, genes previously shown to be differentially regulated during lactation, provide validation for the microarray analysis. New genes identified as being differentially expressed include those related to neurotransmission, growth factors, signal transduction and structure remodeling. These data identify new genes in ARH/VMH that may play an important role in the adaptations of lactation related to hyperphagia, milk production and the suppression of cyclic reproductive function, and may contribute to elucidating a framework for integrating changes in energy intake with the regulation of reproductive function during lactation.