The effects of osmotic stimulation on Fos expression and cell size increase in the supraoptic nucleus were evaluated in intact, sham-operated, and AV3V-lesioned rats. Fos-positive neurons were found mainly in the AV3V regions and the hypothalamic magnocellular neurons in the forebrain in dehydrated intact rats. Intraperitoneal injection of hypertonic saline and chronic dehydration induced a significant increase in number of Fos-positive neurons in the supraoptic nucleus of intact and sham-operated rats. AV3V lesions completely abolished the expression of Fos in SON neurons of rats that were intraperitoneally injected with hypertonic saline and were chronically dehydrated. Chronic dehydration increased significantly cell size of the OXT and AVP magnocelluar neurons in intact and sham-operated rats. However, there was no increase in cell size of those in the AV3V-lesioned rats. These results demonstrate that neural input derived from AV3V regions plays a significant role in causing Fos expression and structural changes such as cell size increase in the hypothalamic magnocellular neurons with osmotic stimulation.