The decrease in NO production was found to correlate well with a decrease in inducible nitric oxide synthase (iNOS) mRNA expression as demonstrated by Northern blot analysis and quantitative RT-PCR. Since the promoter in iNOS gene contains binding motifs for NF-κB/Rel, NF-IL6, and Oct which appear to be important for LPS-mediated iNOS induction, the effects of DEX on the activation of these transcription factors were examined. Treatment of DEX to RAW 264.7 cells induced a dose-related inhibition of NF-κB/Rel in chloramphenicol acetyltransferase activity, while NF-IL6 or Oct activation was not affected by DEX. Treatment of RAW 264.7 cells with DEX inhibited DNA binding of NF-κB/Rel proteins to their cognate DNA site as measured by electrophoretic mobility shift assay. In addition, DEX treatment caused a significant reduction in nuclear c-rel, p65, and p50 protein contents, and these decreases were paralleled by the accumulation of cytoplasmic c-rel, p65, and p50. These results suggest that DEX may inhibit iNOS gene expression by a mechanism involving the blockade of LPS-induced nuclear translocation of NF-κB/Rel.