1α,25(OH)2-20-epi-D3, a synthetic analog of 1α,25(OH)2D3, differs from the natural hormone, by a simple modification in the stereochemistry of the methyl group at carbon-20 of the side chain. Previous studies have shown that the activity of the analog in inhibiting proliferation and inducing differentiation of leukemic cells is several fold greater than that of 1α,25(OH)2D3. At present, the various mechanisms responsible for the enhanced biological activities of the analog have not been clearly elucidated. In one study it is shown that the 20-epi analog induces a conformation of the vitamin D receptor(VDR) that is distinct from that induced by 1α,25(OH)2D3. The modified conformation of the VDR enhances dimerization of VDR with the retinoid X receptor resulting in increased transactivation activity. It is now established that the enhanced biological activities of synthetic analogs of 1α,25(OH)2D3 also depend on their stability and the rate at which they are metabolized. Therefore, in our present study we compared the metabolism of 1α,25(OH)2-20-epi-D3 with that of 1α,25(OH)2D3 in the isolated perfused rat kidney. The metabolism studies reveal that there is a partial block in the further metabolism of the analog, resulting in accumulation of the intermediary metabolite, 1α,25(OH)2-20-epi-24-oxo-D3 and its precursor, 1α,24,25(OH)3-20-epi-D3. We determined that 1α,25(OH)2-20-epi-24-oxo-D3 also induces a conformation of the VDR similar to that induced by the parent analog. Furthermore, this intermediary metabolite is nearly as potent as the parent analog at inducing transactivation of a gene construct containing the human osteocalcin vitamin D-responsive element linked to the thymidine kinase promoter and growth hormone reporter gene. Thus, we conclude that 1α,25(OH)2-20-epi-D3 is metabolically stable and the stability is at the level of its intermediary metabolites, which in turn contribute significantly to the overall biological activity of the analog.