Nitric oxide (·NO) is a short-lived mediator that can be induced by different cytokines and lipopolysaccharide (LPS) in a variety of cell types and produces many physiological and metabolic changes in target cells. In the current study, we show that a combination of cytokines, LPS, and zymosan- activated serum (ZAS; called for convenience cytomix Z) induces production of high concentrations of the NO oxidation products nitrite (NO2/-) and nitrate (NO3/-) by cultured rat fetal lung epithelial type II cells in a time-dependent fashion. Interferon-γ and tumor necrosis factor-α alone did not significantly affect ·NO synthesis, whereas ZAS, LPS, and interleukin- 1β caused only a modest increase in formation of ·NO oxidation products. Production of NO2/- and NO3/- was inhibited by N(G)-monomethyl-L-arginine and cycloheximide. After exposure of these cells to a combination of the above cytokines, Escherichia coli LPS, and ZAS (cytomix Z), enhanced inducible nitric oxide synthase (iNOS) expression was indicated by an elevation in steady-state mRNA specific for iNOS (via Northern blot analysis) and increased immunofluorescence for iNOS after cell permeabilization, incubation with anti-iNOS antibody, and treatment with Cy3.18-conjugated rabbit-specific antibody. The extent of inflammatory mediator-induced ·NO production by alveolar epithelium, which exceeds that of other lung cell types, reveals new insight into mechanisms of pulmonary host defense and pathways of free radical-mediated lung injury.