The objectives of this investigation were 1) to report that pulmonary surfactant inhibits lipopolysaccharide (LPS)-induced nitric oxide (· NO) production by rat alveolar macrophages, 2) to study possible mechanisms for this effect, and 3) to determine which surfactant component(s) is responsible. · NO produced by the cells in response to LPS is due to an inducible · NO synthase (iNOS). Surfactant inhibits LPS-induced · NO formation in a concentration-dependent manner; · NO production is inhibited by ~50 and ~75% at surfactant levels of 100 and 200 µg phospholipid/ml, respectively. The inhibition is not due to surfactant interference with the interaction of LPS with the cells or to disruption of the formation of iNOS mRNA. Also, surfactant does not seem to reduce · NO formation by directly affecting iNOS activity or by acting as an antioxidant or radical scavenger. However, in the presence of surfactant, there is an ~80% reduction in the amount of LPS-induced iNOS protein in the cells. LPS-induced · NO production is inhibited by Survanta, a surfactant preparation used in replacement therapy, as well as by natural surfactant. · NO formation is not affected by the major lipid components of surfactant or by two surfactant-associated proteins, surfactant protein (SP) A or SP-C. However, the hydrophobic SP-B inhibits · NO formation in a concentration-dependent manner; · NO production is inhibited by ~50 and ~90% at SP-B levels of 1-2 and 10 µg/ml, respectively. These results show that lung surfactant inhibits LPS-induced · NO production by alveolar macrophages, that the effect is due to a reduction in iNOS protein levels, and that the surfactant component responsible for the reduction is SP-B.