1α,25-dihydroxy-16-ene vitamin D3(1α,25(OH)2-16-ene-D3), a novel synthetic analog of vitamin D3, has been shown to be a potent antileukemic agent with low potential to cause hypercalcemia (Jung et al., Leukemia Research 18:453, 1994). In a recent in vivo study we reported that 1α,25(OH)2-24-oxo-16-ene-D3 an intermediary metabolite of 1α,25(OH)2-16-ene-D3, exerted immunosuppressive activity equal to its parent, without causing hypercalcemia (Lemire et al.,Endocrinology 135:2818, 1994). The present in vitro study was performed in a human myeloid leukemic cell line (RWLeu-4) to further understand the critical role of the 1α,25(OH)2-24-oxo-16-ene-D3 in generating the unique biological activities exerted by its parent analog. The target tissue metabolism studies in RWLeu-4 cells indicated that 1α,25(OH)2-24-oxo-16-ene-D3 was the major intermediary metabolite of the analog and its concentration exceeded the concentration of the unmetabolized analog itself. Biological activity studies indicated that both the analog and its metabolite were equipotent in their ability to inhibit cell growth and promote differentiation of RWLeu-4 cells and this activity was several fold more potent than the hormone. In addition, the genomic activity of each vitamin D compound was assessed in rat osteosarcoma cells (ROS 17/2.8) by measuring its ability to transactivate a gene construct containing the vitamin D response element (VDRE) of the osteocalcin gene linked to a growth hormone reporter gene. In these studies, both the analog and its metabolite exhibited equal transactivation activity which was several fold greater than that exhibited by the hormone itself. In summary, our results indicate that the production of 1α,25(OH)2-24-oxo-16-ene-D3 as a stable intermediary metabolite in RWLeu-4 cells contributes significantly to the overall biological activity of 1α,25(OH)2-16-ene-D3. Thus, target tissue metabolism of certain vitamin D analogs may result in the intracellular production and accumulation of one or more stable and active intermediary metabolites, which in turn may play a critical role in the final expression of the unique spectrum of biological activities generated by those analogs.