The regulation of nitric oxide synthase 2 (NOS2) in airway epithelial cells plays a key role in the innate host response to a wide variety of microbial agents and also participates in the generation of pathologic airway inflammation. Among the important signalling cascades that direct NOS2 gene expression are nuclear factor kappaB (NFkappaB) and interferon-gamma (IFNgamma)/signal transducer and activator of transcription 1 (STAT-1). Previous studies suggest activator protein-1 (AP-1), in particular c-Fos component of AP-1, influences NOS2 expression. We investigated the effect of c-Fos modulation using RNA interference siRNA on NOS2 gene expression. A549 cells stably transfected with a plasmid overexpressing a c-Fos siRNA construct (FOSi) resulted in a decrease of NOS2 protein inducibility by IFN gamma. In contrast, classical IFN gamma inducible signal transduction pathways interferon regulated factor-1 (IRF-1) and pSTAT-1 were activated at a similar magnitude in FOSi and control cells. DNA-protein binding assays showed that c-Fos binding was present in wild type cells, but reduced in FOSi clones. FOSi clones had activation of NFkappaB detectable by DNA-protein binding assays, which may have contributed to a decrease of NOS2 expression. Overall, these studies indicate that c-Fos is a requisite and specific component for inducible NOS2 expression.