The brain contains an intrinsic vasopressin fiber system the function of which is unknown. It has been demonstrated recently that astrocytes express high levels of a water channel, aquaporin-4 (AQP4). Because vasopressin is known to regulate aquaporin expression and translocation in kidney collecting ducts and thereby control water reabsorption, we hypothesized that vasopressin might serve a similar function in the brain. By recording intrinsic optical signals in an acute cortical slice preparation we showed that evoked neuronal activity generates a radial water flux in the neocortex. The rapid onset and high capacity of this flux suggest that it is mediated through the AQP4-containing astrocytic syncytium that spans the entire thickness of the neocortical mantle. Vasopressin and vasopressin receptor V1a agonists were found to facilitate this flux. V1a antagonists blocked the facilitatory effect of vasopressin and reduced the water flux even in the absence of any exogenous agonist. V2 agonists or antagonists had no effect. These data suggest that vasopressin and V1a receptors play a crucial role in the regulation of brain water and ion homeostasis, most probably by modulating aquaporin-mediated water flux through astrocyte plasma membranes.