Carbon monoxide (CO) is currently being evaluated as a therapeutic modality in the treatment of patients with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). No study has assessed the effects of CO on transepithelial ion transport and alveolar fluid reabsorption, two key aspects of alveolo-capillary barrier function that are perturbed in ALI/ARDS. Both CO gas (250 ppm) and CO donated by the CO donor, CORM-3 (100 mM in epithelial lining fluid), applied to healthy isolated, ventilated and perfused rabbit lungs, significantly blocked ²²Na⁺ clearance from the alveolar compartment, and blocked alveolar fluid reabsorption after fluid challenge. Apical application of two CO donors, CORM-3 or CORM-A1 (100 mM), irreversibly inhibited amiloride-sensitive short-circuit currents in H441 human bronchiolar epithelial cells and primary rat alveolar type II cells, by up to 40%. Using a nystatin permabilization approach, the CO effect was localized to amiloride-sensitive channels on the apical surface. This effect was abolished by hemoglobin, a scavenger of CO, and was not observed when inactive forms of the CO-donors were employed. Neither membrane-permeable 8Br-cGMP nor soluble guanylate cyclase inhibitors (methylene blue and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) or an inhibitor of actin mobility (phalloidin oleate) blocked the effects of CO. However, the amiloride affinity of H441 cells was reduced after CO exposure. Furthermore, with the histidine-modifying agent DEPC (diethyl-pyrocarbonate) the effect of CO could be mimicked and abolished, indicating that histidines are important in the CO-mediated channel inhibition. These data indicate that CO rapidly inhibits sodium absorption across the airway epithelium by cGMP- and actin mobility-independent mechanisms, which may rely on critical histidine residues in amiloride-sensitive channels or associated regulatory proteins on the apical surface of lung epithelial cells. Therefore the use of CO as a therapeutic tool in the treatment of ALI/ARDS should be carefully examined with respect to alveolar fluid clearance and the formation/resolution of pulmonary edema.