The effect of prolonged osmotic stimulation on the oxytocin (OT) mRNA levels of OT-producing neurons was investigated in separate hypothalamic nuclei of the rat. After drinking 2% NaCl for 2 weeks, a 2-fold increase in the OT mRNA content was found by Northern blot analysis of microdissected supraoptic nucleus (SON) and paraventricular nucleus (PVN). The same samples showed a similar change in vasopressin (VP) mRNA levels. This treatment resulted in a 70% depletion of both OT and VP from the neurointermediate lobe of the pituitary gland and a marked increase in OT and VP plasma levels. Analysis of the OT mRNA concentration by quantitative in situ hybridization showed that the SON, PVN, and anterior commissural nucleus responded similarly, with a 1.5-fold increase in OT mRNA after 7 days of osmotic stimulation. These results demonstrate that OT gene expression in the magnocellular neurons of the SON, PVN, and anterior commissural nucleus is sensitive to this osmotic stimulus, as is the VP gene of magnocellular neurons in the SON and PVN. The responsiveness of OT-producing neurons may point to a role for OT in the regulation of water and electrolyte balance.