In oxygen-sensitive excitable cells, responses to hypoxia are initiated by membrane depolarization due to closing of the K channels that is thought to be mediated by a decrease in reactive oxygen species (ROS). Because the mechanisms of hypoxic inhibition of ion transport of alveolar epithelial cells (Planes C, Friedlander G, Loiseau A, Amiel C, and Clerici C. Am J Physiol Lung Cell Mol Physiol 271: L70–L78, 1996; Mairbäurl H, Wodopia R, Eckes S, Schulz S, and Bärtsch P. Am J Physiol Lung Cell Mol Physiol 273: L797–L806, 1997) are not yet understood, we tested the possible involvement of a hypoxia-induced change in ROS that might control transport activity. Transport was measured as ⁸⁶Rb and²²Na uptake in A549 cells exposed to normoxia, hyperoxia, or hypoxia together with ROS donors and scavengers. H₂O₂ < 1 mM did not affect transport, whereas 1 mM H₂O₂ activated²²Na uptake (+200%) but inhibited ⁸⁶Rb uptake (−30%). Also hyperoxia, aminotriazole plus menadione, and diethyldithiocarbamate inhibited ⁸⁶Rb uptake.N-acetyl-l-cysteine, diphenyleneiodonium, and tetramethylpiperidine-N-oxyl, used to reduce ROS, inhibited⁸⁶Rb uptake, thus mimicking the hypoxic effects, whereas deferoxamine, superoxide dismutase, and catalase were ineffective. Also, hypoxic effects on ion transport were not prevented in the presence of H₂O₂, diethyldithiocarbamate, andN-acetyl-l-cysteine. These results indicate that ion transport of A549 cells is significantly affected by decreasing or increasing cellular ROS levels and that it is possible that certain species of ROS might mediate the hypoxic effects on ion transport of alveolar epithelial cells.