TTF-1 is a member of the Nkx family of homeodomain genes required for morphogenesis of the hypothalamus. Whether TTF-1, or other Nkx genes, contributes to regulating differentiated hypothalamic functions is not known. We now report that postnatal hypothalamic TTF-1 expression is developmentally regulated and associated with the neuroendocrine process of female sexual development. Lesions of the hypothalamus that cause sexual precocity transiently activate neuronal TTF-1 expression near the lesion site. In intact animals, hypothalamic TTF-1 mRNA content also increases transiently, preceding the initiation of puberty. Postnatal expression of the TTF-1 gene was limited to subsets of hypothalamic neurons, including LHRH neurons, which control sexual maturation, and preproenkephalinergic neurons of the lateroventromedial nucleus of the basal hypothalamus, which restrain sexual maturation and facilitate reproductive behavior. TTF-1 mRNA was also detected in astrocytes of the median eminence and ependymal/subependymal cells of the third ventricle, where it colocalized with erbB-2, a receptor involved in facilitating sexual development. TTF-1 binds to and transactivates the erbB-2 and LHRH promoters, but represses transcription of the preproenkephalin gene. The singular increase in hypothalamic TTF-1 gene expression that precedes the initiation of puberty, its highly specific pattern of cellular expression, and its transcriptional actions on genes directly involved in neuroendocrine reproductive regulation suggest that TTF-1 may represent one of the controlling factors that set in motion early events underlying the central activation of mammalian puberty.