Phenolic antioxidants inhibit the induction of inflammatory cytokines by inflammatory stimuli. Here, we analyzed the mechanism by which the antioxidants inhibit LPS-induced expression of tumor necrosis factor (TNF) in macrophages. Hydroquinone and tert-butyl hydroquinone, prototypes of phenolic antioxidants, block lipopolysaccharide (LPS)-induced transcription of TNF and a nuclear factor (NF)-B mediated reporter gene expression, suggesting NF-B as a target in the inhibition. Analyses of the NF-B activation pathway revealed that the antioxidants do not inhibit LPS-induced activation of the IB kinase activity, degradation of IB, or translocation of activated NF-B into the nucleus, but they do block the formation of NF-B/DNA binding complexes. In vitro experiments showed that the antioxidants do not directly interfere with DNA binding of NF-B. Structure-activity analyses suggest that inhibition of NF-B function involves the redox cycling property of the antioxidants. These findings implicate a redox-sensitive factor important for the binding of NF-B to its DNA recognition sequence as a target molecule in the inhibition of NF-B function and inflammatory cytokine expression by phenolic antioxidants.