The hypothalamo-neurohypophyseal system (HNS) mediates neuroendocrine responses to dehydration through the action of the antidiuretic hormone vasopressin (VP). VP is synthesized as part of a prepropeptide in magnocellular neurons of the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus. This precursor is processed during transport to axon terminals in the posterior pituitary gland, in which biologically active VP is stored until mobilized for secretion by electrical activity evoked by osmotic cues. During release, VP travels through the blood stream to specific receptor targets located in the kidney in which it increases the permeability of the collecting ducts to water, reducing the renal excretion of water, thus promoting water conservation. The HNS undergoes a dramatic function-related plasticity during dehydration. We hypothesize that alterations in steady-state protein levels might be partially responsible for this remodeling. We investigated dehydration-induced changes in the SON and pituitary neurointermediate lobe (NIL) proteomes using two-dimensional fluorescence difference gel electrophoresis. Seventy proteins were altered by dehydration, including 45 in the NIL and 25 in the SON. Using matrix-assisted laser desorption/ionization mass spectrometry, we identified six proteins in the NIL (four down, two up) and nine proteins in the SON (four up, five down) that are regulated as a consequence of chronic dehydration. Results for five of these proteins, namely Hsp1alpha (heat shock protein 1alpha), NAP22 (neuronal axonal membrane protein 22), GRP58 (58 kDa glucose regulated protein), calretinin, and ProSAAS (proprotein convertase subtilisin/kexin type 1 inhibitor), have been confirmed using independent methods such as semiquantitative Western blotting, two-dimensional Western blotting, enzyme-linked immunoassay, and immunohistochemistry. These proteins may have roles in regulating and effecting HNS remodeling.