Free radicals, or reactive oxygen species (ROS), are highly reactive byproducts of oxygen degradation. They are well known for their cellular toxicity, but few studies have analyzed their potential role in homeostatic processes. We investigated ROS production and function during the arginine vasopressin (AVP) hypothalamic response to hyperosmolarity. Six-week-old male C3H/HeJ mice were subjected to salt loading for 2 or 8 d. The osmotic axis was progressively activated and reached a new steady-state status at 8 d as demonstrated by monitoring of plasmatic osmolality and c-Fos and AVP expression in the supraoptic nucleus (SON). Free radicals, visualized by dihydroethidine staining and measured by 2′-7′dichlorofluorescein diacetate assays, were detected after 2 d of salt loading. The activity and expression of superoxide dismutase 2 and catalase were concomitantly up-regulated in the SON, suggesting that free radicals are detoxified by endogenous antioxidant systems, thereby avoiding their deleterious effects. The early phase of the osmoregulatory response has been investigated using an acute hyperosmotic model; free radicals were produced 45 min after an ip injection of 1.5 M NaCl. This was followed by an increase in c-Fos and AVP expression and an increase in superoxide dismutase 2 and catalase activities. α-Lipoic acid, a ROS scavenger, administrated during the 3 d before the hypertonic ip injection, abolished the increase of AVP. These findings establish that hyperosmolarity causes ROS production in the SON, which is essential for AVP increase. This demonstrates the importance of free radicals as physiological signaling molecules in the regulation of body-fluid balance.