ABSTRACT Tumor necrosis factor-alpha (TNF-alpha) exerts pleiotropic effects on thyroid follicular cells. However, the intracellular signaling pathway for the TNF-alpha action has not been well elucidated. The present study examined the effects of TNF-alpha on the activation of nuclear factor-kappa B (NF-kappaB) and on the expression of interleukin (IL)-6 gene in rat thyroid FRTL-5 cells. The treatment of the cells with TNF-alpha resulted in the nuclear translocation of p65-p50 heterodimer as well as p50-p50 homodimer NF-kappaBs. The treatment with the antioxidants 20 mM N-acetyl-L-cysteine (NAC) and 10 microM pyrrolidine dithiocarbamate (PDTC) inhibited the TNF-alpha-dependent activation of p65-p50 heterodimer but not the p50-p50 homodimer, indicating that generation of oxidants is required for the activation of the heterodimer NF-kappaB. When the plasmid containing the multimerized NF-kappaB sites upstream of a luciferase reporter gene was transfected into FRTL-5 cells, the treatment with NAC or PDTC prevented the TNF-alpha-dependent increase in the luciferase activities, indicating that the p65-p50 heterodimer is a transcriptionally active NF-kappaB. Accordingly, the TNF-alpha-dependent increase in IL-6 messenger RNA and in secretion of the protein was prevented by the treatment with NAC. These results strongly suggest that TNF-alpha increases the IL-6 gene expression through the activation of NF-kappaB in the thyroid cells, and that antioxidants suppress the TNF-alpha-dependent IL-6 expression by inhibiting the activation of the transcriptionally active NF-kappaB.