We have evaluated seven recently synthesized vitamin D3 analogs for their abilities to inhibit clonal growth of leukemic cells, to induce leukemic cell differentiation, to stimulate clonal growth of normal myeloid committed stem cells, and to transactivate a reporter gene having a 1,25(OH)2D3 response element (VDRE). The 1,25(OH)2-20-epi-D3 showed extraordinary activity; at 10(-11) mol/L it inhibited clonal growth of 87% of HL-60 myeloblast cells, 60% of S-LB1 cells (human T-cell lymphotropic virus type 1 [HTLV-1]-immortalized human T-lymphocyte cell line) and 50% of leukemic clonogenic cells (colony-forming unit-leukemia) obtained from patients with acute myelogenous leukemia. No effect of either 1,25(OH)2D3 or 1,25(OH)2-20-epi-D3 was observed on the clonal proliferation of an HTLV-1-immortalized human T-lymphocyte cell line (Ab-VDR) having nonfunctional 1,25 (OH)2D3 cellular receptors (VDR). The abilities of 1,25(OH)2-20-epi-D3 to induce differentiation of HL-60 cells, as measured by generation of superoxide and nonspecific esterase production, was less than its antiproliferative activities. This analog stimulated colony-forming unit-granulocyte-macrophage growth from normal human bone marrow. To gain insights into the remarkable antileukemic activities of 1,25(OH)2-20-epi-D3, we examined its ability to enter HL-60 cells, bind to the VDR, and interact with a transfected VDRE attached upstream of a TK promoter-driven reporter gene (chloramphenicol acetyl transferase [CAT]). The 1,25(OH)2-20-epi-D3 potently increased CAT activity (> 16-fold, as compared with cells transfected with control receptor having no VDRE); paradoxically, 1,25(OH)2-20-epi-D3 was of equal potency to 1,25(OH)2D3 in transactivating the VDRE-containing reporter gene, even though the analog had a 1,000-fold greater antileukemic effect as compared with 1,25(OH)2D3. In summary, we have identified an extremely potent 1,25(OH)2D3 analog with antiproliferative and differentiating effects on leukemic cells and that may be clinically useful. This analog appears to generate biologic responses via the classical VDR pathway, but further studies are required to elucidate the mechanism by which this analog produces its prominent activities.