The hypothalamo-neurohypophyseal system (HNS) mediates neuroendocrine responses to dehydration through the action of the antidiuretic hormone vasopressin (VP). VP is synthesised as part of a prepropeptide in magnocellular neurons (MCNs) of the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN). This precursor is processed during transport to axon terminals in the posterior pituitary gland (PP), where biologically active VP is stored until mobilised for secretion by electrical activity evoked by osmotic cues. Upon release, VP travels through the blood stream to specific receptor targets located in the kidney where 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 hypothesise that alterations in steady-state protein levels might be partially responsible for this remodelling. We have investigated dehydration-induced changes in the SON and pituitary neurointermediate lobe (NIL) proteomes using 2-dimensional (2D) Fluorescence Difference Gel Electrophoresis (DIGE). Seventy proteins were altered by dehydration, including 45 in the NIL and 25 in the SON. Using MALDI mass spectrometry (MS), we have identified 6 proteins in the NIL (4 down, 2 up) and 9 proteins in the SON (4 up, 5 down) that are regulated as a consequence of chronic dehydration. Results for 5 of these proteins, namely Hsp1, NAP22, GRP58, calretinin and ProSAAS, have been confirmed using independent methods such as semi-quantitative Western blotting, 2D Western blotting, enzyme-linked immunoassay (EIA) and immunohistochemistry. These proteins may have roles in regulating and effecting HNS remodelling.