We investigated the mechanisms of endogenous nitric oxide (NO) modulation of lung sodium (Na+) transport. C57BL/6 mice injected intraperitoneally with the specific inducible NO synthase (INOS) inhibitor 1400W (10 mg/kg every 8 h for 72 h) exhibited decreased alveolar nitrite levels and Na+-dependent amiloride-sensitive alveolar fluid clearance as compared with mice injected with vehicle. Similarly, pretreatment of mouse tracheal epithelial cells with 1400W abolished the inhibitory effects of amiloride on their Na+ short circuit currents. On the other hand, mouse tracheal epithelial cells pretreated with 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one, a specific inhibitor of guanylate cyclase, had lower levels of cGMP, but normal values of amiloride-sensitive Na+ currents. Amiloride also inhibited whole-cell Na+ currents across A549 cells treated with vehicle (K1 = 249 nM), but had no effect in A549 cells treated with 1400W. Western blotting studies showed significantly lower levels of a and γENaC in lung tissues and alveolar type II (ATII) cells from INOS-/- as well as iNOS+/+ mice treated with 1400W, as compared with the corresponding values from vehicle-treated INOS+/+ mice. Similar values for ratios of α, β, and γenac to gapdh were obtained by real-time polymerase chain reaction for iNOS+/+ mice and INOS-/- mice. We concluded that NO derived from iNOS under basal conditions is necessary for amiloride-sensitive Na+ transport across lung epithelial cells and modulates the amount of α and γENaC via posttranscriptional, cGMP-independent mechanisms.