Expression of inflammatory cytokines is regulated by transcriptional and posttranscriptional mechanisms. We previously showed that NADPH oxidase-derived superoxide induces inflammatory mediators in response to tumor necrosis factor α (TNF-α) and lipopolysaccharide (LPS). In this study, we examined the role of endothelial NADPH oxidase in the regulation of mRNA stability of three inflammatory mediators: interleukin (IL) 8, IL-6, and intercellular adhesion molecule 1 (ICAM-1). Tumor necrosis factor α increased mRNA stability of ICAM-1, IL-8, and IL-6 by a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism, but this did not involve NADPH oxidase. Surprisingly, whereas LPS treatment alone did not alter stability of these molecules, the antioxidant N-acetyl-L-cysteine; the flavine inhibitor diphenylene iodonium; short interfering RNA against Nox2, Nox4; and the p22(phox) subunit of NADPH oxidase all enhanced IL-8 mRNA stability in LPS-treated cells, indicating that LPS induced destabilization through NADPH oxidase. This occurred by a mechanism that involved extracellular signal-regulated kinase 1/2, p38 MAPK, and the mRNA-destabilizing factor tristetraprolin. On the other hand, N-acetyl-L-cysteine decreased mRNA stability of ICAM-1 and IL-6 in LPS-treated cells and IL-6 and ICAM-1 in TNF-α-treated cells. In conclusion, NADPH oxidase contributes to destabilization of IL-8 mRNA stability and propose a model for the complex underlying mechanism, which is dependent upon agonist (LPS vs. TNF-α) and target molecule (IL-8 vs. IL-6 and ICAM-1) and involves tristetraprolin, p38, and extracellular signal-regulated kinase 1/2 MAPK.