A decrease in serum sodium ion concentration below 135 mmol L-1 is usually accompanied by a decrease in plasma osmolality (hypoosmotic hyponatremia) and leads to the disorder of intracranial homeostasis mainly due to cellular swelling. Recently, using an in vitro model of hypoosmotic hyponatremia, we have found that a decrease in sodium ion concentration in the perfusate to 121 mmol L-1 relaxes the isolated rat middle cerebral artery (MCA). The aim of the present study was to explore the mechanism responsible for this relaxation. Isolated, pressurized, and perfused MCAs placed in a vessel chamber were subjected to a decrease in sodium ion concentration to 121 mmol L-1. Changes in the diameter of the vessels were monitored with a video camera. The removal of the endothelium and inhibition of nitric oxide-dependent signaling or the reverse mode sodium-calcium exchanger (NCX) were used to study the mechanism of the dilation of the vessel during hyponatremia. The dilation of the MCA (19 ± 5%, p < 0.005) in a low-sodium buffer was absent after removal of the endothelium or administration of the inhibitor of the reverse mode of sodium-calcium exchange and was reversed to constriction after the inhibition of nitric oxide (NO)/cGMP signaling. The dilation of the middle cerebral artery of the rat in a 121 mmol L-1 Na+ buffer depends on NO signaling and reverse mode of sodium-calcium exchange. These results suggest that constriction of large cerebral arteries with impaired NO-dependent signaling may be observed in response to hypoosmotic hyponatremia.