Objective：To investigate the effect of Na^＋,K^＋-ATPase on the increase of cytosolic Ca^2＋ level induced by hypoxia in rat cortical neurons. Methods： Using confocal laser scanning microscope and video based motion edge detection system, we measured the intraeellular Ca^2＋ concentration （[Ca2＋]i ） and Na^＋ concentration （[Na^-]i ） in cortical neurons exposed to hypoxia for different time periods and at presence of different concentrations of dihydroouabain （DHO, an inhibitor of Na^＋,K^＋-ATPase ）. We also observed the effect ofNa^＋,K^＋-ATPase on the increase of cytosolic Ca^2＋ level induced by hypoxia in rat cortical neurons. Results： DHO （10^-9-10^-3mol/L） dose-dependently and hypoxia（4-20 min） time-dependently increased the levels of [Ca^2＋] and [Na^＋] in cultured cortical neurons. [Ca^2＋]i and [Na^＋]i increased significantly 4 min after cortical neurons were exposed to hypoxia, and addition of DHO （ 10^-3mol/L） further increased their concentrations. While 15 min after hypoxia exposure, different concentrations of DHO did not further increase [Ca^2＋] and [Na^＋]i We also found that hypoxia exposure did not enhance DHO pretreatment-induced [Ca^2＋]i increase. Couclusion： Inhibition of Na^＋,K^＋-ATPase activity is one of the mechanisms responsible for hypoxia-induced elevation of [Ca2＋]i in cortical neurons.