Phenolic antioxidants exhibit anti-inflammatory activity in protection against chemical toxicity and cancer. To investigate the molecular mechanism of anti-inflammation, we analyzed the regulation of tumor necrosis factor alpha (TNF-alpha) expression in macrophages, a key step in inflammation, by the antioxidants. Whereas lipopolysaccharide (LPS), an inflammatory inducer, stimulates rapid synthesis of TNF-alpha protein, phenolic antioxidants, exemplified by tert-butyl hydroquinone and 1,4-dihydroquinone, block LPS-induced production of TNF-alpha protein in a time- and dose-dependent manner. Inhibition of TNF-alpha induction correlates with the capacity of the antioxidants to undergo oxidation-reduction cycling, implicating oxidative signaling in the inhibition. The antioxidants blocked LPS-induced increase of the steady-state mRNA of TNF-alpha but did not affect the half-life of the mRNA. Electrophoretic mobility shift assay reveals a total inhibition of LPS-induced formation of nuclear factor kappaB.DNA binding complexes by phenolic antioxidants. Finally, 1,4-dihydroquinone blocks the induction of TNF-alpha target genes interleukin 1beta and interleukin 6 at both mRNA and protein levels. Our findings demonstrate that phenolic antioxidants potently inhibit signal-induced TNF-alpha transcription and suggest a mechanism of anti-inflammation by the antioxidants through control of cytokine induction during inflammation.