We examined the mechanisms by which nitric oxide (·NO) decreased vectorial Na+ transport across confluent monolayers of rat alveolar type II (ATII) cells grown on permeable supports. Amiloride (10 μM) applied to the apical side of monolayers inhibited ~90% of the equivalent (I(eq)) and the short-circuit (I(sc)) current, with an half-maximal inhibitory concentration (IC50) of 0.85 μM, indicating that Na+ entry into ATII cells occurred through amiloride-sensitive Na+ channels. ·NO generated by spermine NONOate and papa NONOate added to both sides of the monolayers decreased I(eq) and increased transepithelial resistance in a concentration-dependent fashion (IC50 = 0.4 μM ·NO). These changes were prevented or reversed by addition of oxyhemoglobin (50 μM). Incubation of ATII monolayers with 8- bromoguanosine 3',5'-cyclic monophosphate (400 μM) had no effect on transepithelial Na+ transport. When the basolateral membranes of ATII cells were permeabilized with amphotericin B (10 μM) in the presence of a mucosal- to-serosal Na+ gradient (145:25 mM), ·NO (generated by 100 μM papa NONOate) inhibited ~60% of the amiloride-sensitive I(sc). In addition, after permeabilization of the apical membranes, ·NO inhibited the I(sc) [a measure of Na+-K+-adenosinetriphosphatase (ATPase) activity] by ~60%. We concluded that ·NO at noncytotoxic concentrations decreased Na+ absorption across cultured ATII monolayers by inhibiting both the amiloride-sensitive Na+ channels and Na+-K+-ATPase through guanosine 3',5'-cyclic monophosphate- independent mechanisms.