Nitric oxide (NO) is implicated in the pathogenesis of lung inflammation and edema. In this study, the effects of nitric oxide (NO)-donors on membrane water permeability and cell surface expression of aquaporin-5 (AQP5) in mouse lung epithelial cells were examined. NO-donors, GSNO and NOC-18 decreased cell surface expression of AQP5, concentration- and time-dependently, whereas they did not affect the amount of AQP5 in whole cell lysates. The membrane water permeability of cells was also decreased by treatment with NO-donors. The decrease in cell surface AQP5 by NO was abolished by simultaneous treatment with methyl-beta-cyclodextrin, but not with ODQ, an inhibitor of the cGMP-dependent pathway. In addition, immunocytochemistry with anti-AQP5 indicated that NO changed AQP5 localization from the plasma membrane to the intracellular fraction. These data indicate that NO stimulates AQP5 internalization from the plasma membrane through a cGMP-independent mechanism, and decreases membrane water permeability.