Responsiveness to 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] may be diminished in osteoporosis and inflammatory arthritis. The inflammatory cytokine tumor necrosis factor- (TNF-) is produced in excess in these disorders and has been shown to decrease osteoblast transcriptional responsiveness to vitamin D and to inhibit the binding of the vitamin D receptor (VDR) and its nuclear partner the retinoid X receptor (RXR) to DNA. Previous studies have shown that a vitamin D (VDRE) or retinoid X DNA response element (RXRE) is sufficient to confer TNF- inhibition of vitamin D or retinoid-stimulated transcription in the absence of known TNF--responsive DNA sequences. We tested the hypothesis that the TNF--stimulated transcription factor nuclear factor (NF)-B could, in part, mediate TNF- action by inhibiting the transcriptional potency of the VDR and RXR at their cognate cis regulatory sites. Osteoblastic ROS 17/2.8 cells transfected with a dose of NF-B comparable to that stimulated by TNF- decreased 1,25(OH)₂D₃-stimulated transcription. This inhibitory effect of NF-B was not observed on basal transcription of a heterologous reporter in the absence of the VDRE. The effects of NF-B and TNF- were comparable but not additive. COS-7 cells were cotransfected with reporters under the regulation of VDRE or RXRE along with vectors expressing VDR, RXR, and NF-B nuclear proteins. Reconstituted NF-B and the NF-B subunit p65 alone, but not p50, dose dependently suppressed basal and ligand-stimulated transcription. p65 overexpression completely abrogated enhanced VDRE-mediated transcriptional activity in response to 1,25(OH)₂D₃. Electrophoretic mobility shift experiments did not reveal a direct effect of recombinant NF-B or its individual subunits on the binding of heterodimeric VDR-RXR to DNA. These results suggest that TNF- inhibition of hormone-stimulated transcriptional activation may be mediated by activation of NF-B. In contrast, the inhibitory effect of TNF- on binding of receptors to DNA is unlikely to be mediated by NF-B and is not necessary for inhibition of transcription.