Abstract We have demonstrated that 1α,25-dihydroxyvitamin D3-26,23-lactone analogs, (23S)- and (23R)-25-dehydro-1α-hydroxyvitamin D3-26,23-lactone (TEI-9647, TEI-9648, respectively), inhibit HL-60 cell differentiation induced by 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], but not differentiation caused by all-trans retinoic acid (D. Miura et al., 1999, J. Biol. Chem. 274, 16392). To assess whether the antagonistic actions of TEI-9647 and TEI-9648 in HL-60 cells are related to 1α,25(OH)2D3 breakdown, we investigated their effects on catabolism of 1α,25(OH)2D3. In HL-60 cells, the C-24 but not the C-23 side-chain oxidation pathway of 1α,25(OH)2D3 has been reported. Here we demonstrate that 1α,25(OH)2D3 was metabolized both to 24,25,26,27-tetranor-1α,23-(OH)2D3 and 1α,25(OH)2D3-26,23-lactone; thus HL-60 cells constitutively possess both the 24- and the 23-hydroxylases. Metabolism of 1α,25(OH)2D3 was strongly suppressed by 10−7 M TEI-9647 or 10−6 M TEI-9648. 1α,25(OH)2D3 alone slightly induced 24-hydroxylase gene expression by 8 h with full enhancement by 24–48 h; this induction was inhibited by 10−6 M TEI-9647 and 10−6 M TEI-9648 (86.2 and 31.9%, respectively) 24 h after treatment. However, analogs of TEI-9647 and TEI-9648 without the 25-dehydro functionality induced 24-hydroxylase gene expression. These results indicate that TEI-9647 and TEI-9648 clearly mediate their stereoselective antagonistic actions independent of their actions to block the catabolism of 1α,25(OH)2D3. Therefore, TEI-9647 and TEI-9648 appear to be the first antagonists specific for the nuclear 1α,25(OH)2D3 receptor-mediated genomic actions of 1α,25(OH)2D3 in HL-60 cells.