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 intracellular Ca 2+ concentration (\ i ) and Na + concentration (\ 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 of Na + , K + -ATPase on the increase of cytosolic Ca 2+ level induced by hypoxia in rat cortical neurons. Results: DHO (10 -9 -10 -3 mol/L) dose-dependently and hypoxia(4-20 min) time-dependently increased the levels of \ i and \ i in cultured cortical neurons. \ i and \ i increased significantly 4 min after cortical neurons were exposed to hypoxia, and addition of DHO (10 -3 mol/L) further increased their concentrations. While 15 min after hypoxia exposure, different concentrations of DHO did not further increase \ i and \ i. We also found that hypoxia exposure did not enhance DHO pretreatment-induced \ i increase. Conclusion: Inhibition of Na + , K + -ATPase activity is one of the mechanisms responsible for hypoxia-induced elevation of \ i in cortical neurons.