(23S)-25-dehydro-1alpha-Dihydroxyvitamin D3-26,23-lactone (TEI-9647; MK) has been reported to antagonize the 1alpha,25-dihydroxyvitamin D3 nuclear receptor (VDR)- mediated increase in transcriptional activity. Using a transient transfection system incorporating the osteocalcin VDRE (vitamin D response element) in Cos-1 cells, we found that 20 nM MK antagonizes VDR-mediated transcription by 50% when driven by 1 nM 1alpha,25(OH)2D3. Four analogs of 1alpha,25(OH)2D3, also at 1 nM, were antagonized 25 to 39% by 20 nM MK. However, analogs with 16-ene/23-yne or 20-epi modifications, which have a significantly lower agonist ED50 for the VDR than 1alpha,25(OH)2D3, were antagonized by 20 nM MK only at 100 pM or 10 pM, respectively. One possible mechanism for antagonism is that the 25-dehydro alkene of MK might covalently bind the ligand-binding site of the VDR rendering it inactive. Utilization of a ligand exchange assay, however, demonstrated that MK bound to VDR is freely exchanged with 1alpha,25(OH)2D3 in vitro. These data support the apparent correlation between VDR transcriptional activation by agonists and the effective range of MK antagonism by competition. Furthermore, protease sensitivity analysis of MK bound to VDR indicates the presence of a unique conformational change in the VDR ligand-binding domain, showing a novel doublet of VDR fragments centered at 34 kDa, whereas 1alpha,25(OH)2D3 as a ligand produces only a single 34-kDa fragment. In comparison, the natural metabolite 1alpha,25-dihydroxyvitamin D3-26,23-lactone yields only the 30-kDa fragment that is produced by all ligands to varying degrees. Collectively, these results support that MK is a potent partial antagonist of the VDR for 1alpha,25(OH)2D3 and its analogs when in appropriate excess of the agonist.