Estrogens are the most effective agents available for preventing osteoporosis, and their principal role in bone metabolism is the inhibition of interleukin-6 (IL-6) production in osteoblasts and bone marrow stromal cells. We examined the mechanism of inhibitory effect of estrogens on the 190 bp proximal promoter of the IL-6 gene. Promoter activity induced by transfection of both NF-kB p65 subunit and NF-IL6 was decreased by 45% by estradiol (E₂)-estrogen receptor (ER) complexes. The inhibitory effect of E₂ was also observed on a mutant IL-6 promoter in which the NF-IL6 binding site was disrupted. E₂ repressed the wild-type promoter activity induced by NF-kB p65 subunit alone, but had no effect on that induced by NF-IL6 alone. These findings suggested that estrogens inhibit IL-6 production by interfering with the function of NF-kB rather than that of NF-IL6. The ER mutant, HE19, which does not contain the A B domain, repressed the induction by NF-kB to the same extent as wild-type ER HE0, whereas the effect of C-terminal deletion mutant, HE21, was only marginal. The antiestrogen, 4-hydroxytamoxifen (OHT), had no effect on IL-6 promoter activity, suggesting that E₂-induced conformational change of the hormone binding domain plays an important role in protein-protein interaction between ER and NF-kB. E₂ had no effect on the nuclear translocation of NF-kB, and electrophoretic mobility shift assay showed that the presence of E₂-ER complexes did not affect the ability of NF-kB to bind to specific DNA sequences.