The extent to which endogenously generated nitric oxide alters Na⁺ transport across the mammalian alveolar epithelium in vivo has not been documented. Herein we measured alveolar fluid clearance and nasal potential differences in mice lacking the inducible form of nitric oxide synthase [iNOS; iNOS(−/−)] and their corresponding wild-type controls [iNOS(+/+)]. Alveolar fluid clearance values in iNOS(+/+) and iNOS(−/−) anesthetized mice with normal oxygenation and acid-base balance were ∼30% of instilled fluid/30 min. In both groups of mice, fluid absorption was dependent on vectorial Na⁺ movement. Amiloride (1.5 mM) decreased alveolar fluid clearance in iNOS(+/+) mice by 61%, whereas forskolin (50 μM) increased alveolar fluid clearance by 55% by stimulating amiloride-insensitive pathways. Neither agent altered alveolar fluid clearance in iNOS(−/−) mice. Hyperoxia upregulated iNOS expression in iNOS(+/+) mice and decreased their amiloride-sensitive component of alveolar fluid clearance but had no effect on the corresponding values in iNOS(−/−) mice. Nasal potential difference measurements were consistent with alveolar fluid clearance in that both groups of mice had similar baseline values, which were amiloride sensitive in the iNOS(+/+) but not in the iNOS(−/−) mice. These data suggest that nitric oxide produced by iNOS under basal conditions plays an important role in regulating amiloride-sensitive Na⁺ channels in alveolar and airway epithelia.