We describe a hypothalamus-specific mRNA that encodes preprohypocretin, the putative precursor of a pair of peptides that share substantial amino acid identities with the gut hormone secretin. The hypocretin (Hcrt) protein products are restricted to neuronal cell bodies of the dorsal and lateral hypothalamic areas. The fibers of these neurons are widespread throughout the posterior hypothalamus and project to multiple targets in other areas, including brainstem and thalamus. Hcrt immunoreactivity is associated with large granular vesicles at synapses. One of the Hcrt peptides was excitatory when applied to cultured, synaptically coupled hypothalamic neurons, but not hippocampal neurons. These observations suggest that the hypocretins function within the CNS as neurotransmitters.The hypothalamus acts as a major regulatory center for autonomic and endocrine homeostasis. Structurally, it is a confederation of nuclei that regulate a broad array of physiological and behavioral activities. For some of these activities, particular peptides have been identified as major products of individual nuclei (1). These peptides exert their actions by transport to the pituitary, by entering the general circulation, or by secretion within the CNS. However, the hypothalamus has been implicated in the regulation of activities beyond those for which factors have been identified.We recently used directional tag PCR subtraction to identify 38 rat mRNAs selectively expressed within the hypothalamus (2). Preliminary in situ hybridization studies revealed that one of these, called clone 35 in that work, was expressed exclusively by a bilaterally symmetric structure within the posterior hypothalamus. Here we show that the clone 35 mRNA encodes the precursor of two putative peptides, the hypocretins, that share substantial amino acid identities with each other and with the gut hormone secretin. The Hcrt mRNA, which accumulates primarily after postnatal week 3 and in mouse is a product of a gene on chromosome 11, is restricted to neuronal cell bodies of the dorsal and lateral hypothalamus. Its protein product, visualized immunocytochemically, is sorted into secretory vesicles in fibers that project within the hypothalamus and to other brain areas. At least one of the peptides has neuroexcitatory activity. Cumulatively, these observations suggest that the Hcrt mRNA encodes peptides that act endogenously within the central nervous system as homeostatic regulators. The circuitry revealed by the immunohistochemistry suggests a role in nutritional homeostasis. MATERIALS AND METHODSProduction of Antisera. Antiserum 2050 was generated by coupling the synthetic 17-mer CPTATATALAPRGGSRV to the carrier keyhole limpet hemocyanin with glutaraldehyde and immunizing rabbits as described (3). In Western transfer blots using as target electrophoretically separated proteins from bacteria transformed with the plasmid pRSET B engineered to express preprohcrt, we observed a single prominent immunoreactive band with a migration of Ϸ19 kDa with the hyperi...