Aspirin has been reported to inhibit the activation of nuclear factor-kappaB (NF-kappaB) through stabilization of inhibitor kappaB (IkappaB). This observation led us to investigate the role of aspirin in suppressing the activation of the NF-kappaB-regulated tumor necrosis factor-alpha (TNF-alpha) gene expression in primary macrophages. We now report that therapeutic doses of aspirin suppress lipopolysaccharide-inducible NF-kappaB binding to an NF-kappaB binding site in the TNF-alpha promoter, lipopolysaccharide-induced TNF-alpha mRNA accumulation, and protein secretion. IkappaB is also stabilized under these conditions. The aspirin-initiated stabilization of IkappaB, suppression of induced TNF-alpha mRNA, and NF-kappaB binding to the TNF-alpha promoter are blocked by pretreatment with pertussis toxin. These studies suggest that aspirin may exert significant anti-inflammatory effects by suppressing the production of macrophage-derived inflammatory mediators.