Transepithelial alveolar sodium transport mediated by the amiloride-sensitive epithelial sodium channel (ENaC) constitutes the driving force for removal of fluid from the alveolar space. To define the role of the -ENaC subunit in vivo in the mature lung, we studied a previously established mouse strain harbouring a disruption of the -ENaC gene locus resulting in low levels of -ENaC mRNA expression. Real-time RT-PCR experiments confirmed that -ENaC mRNA levels were decreased by more than 90% in alveolar epithelial cells from homozygous mutant (m/m) mice. -ENaC protein was undetected in lung homogenates from m/m mice by western blotting, but - and -ENaC proteins were increased by 85% and 45% respectively, as compared with wild-type (wt) mice. At baseline, sodium-driven alveolar fluid clearance (AFC) was significantly reduced by 32% in m/m mice. Amiloride at the concentration 1mM inhibited AFC by 75% and 34% in wt and m/m mice respectively, whereas a higher concentration (5mM) induced a 75% inhibition of AFC in both groups. The 2-agonist terbutaline significantly increased AFC in wt but not in m/m mice. These results show that despite the compensatory increase in - and -ENaC protein expression observed in mutant mouse lung, low expression of -ENaC results in a moderate impairment of baseline AFC and in decreased AFC sensitivity to amiloride, suggesting a possible change in the stoichiometry of ENaC channels. Finally, adequate -ENaC expression appears to be required for AFC stimulation by ₂-agonists.