Due to its anti-inflammatory properties, the administration of low doses of carbon monoxide (CO) is emerging as a putative intervention in inflammation-associated lung diseases such as acute lung injury. However, the effects of CO on pulmonary transepithelial sodium absorption and thus, alveolar fluid clearance, are poorly understood. In the present study, administration of either CO gas or the CO donor molecule CORM-3 decreased active sodium clearance in isolated, ventilated and perfused rabbit lungs, leading to increased epithelial lining fluid volume after fluid challenge. Application of CO to cultured airway epithelial cell monolayers (H441 and primary rat alveolar type 2 cells) in Ussing chambers resulted in decreased amiloride-sensitive sodium current, due to impaired activity of amiloride-sensitive sodium channels on the apical membrane. Classically, the epithelial sodium channel (ENaC) is thought to consist of three subunits (a, b and g). Surprisingly, when human lung abgENaC was expressed in Xenopus oocytes, channel activity was increased after CO application. Thus, it was hypothesized that another subunit of ENaC in lung epithelial cells (such as dENaC) could account for the opposite responses to CO observed in the different models. Expression of dENaC in H441 cells was confirmed by western blot. The application of the dENaC antagonist Evans blue also decreased amiloride-sensitive currents in H441 cells, whereas it increased the activity of abgENaC in oocytes. Furthermore, in H441 cells, the effects of Evans blue and CO were not additive, indicating that both act on the same cellular target. Therefore, these data suggest that dENaC might play an important role in modulating the activity of epithelial sodium channels in response to low doses of CO in lung epithelial cells.