Differentiation therapy is being developed as an additional therapeutic option for the treatment of several forms of cancer, including myeloid leukemia. In model systems, the physiologically active form of vitamin D, 1, alpha25-dihydroxyvitamin D3 (1,25D), induces monocytic differentiation of human myeloid cells, but the mechanism is not clear. We report here, the first direct connection between the signal provided by 1,25D and the molecular circuitry known to be involved in monocytic differentiation. Specifically, we show that 1,25D selectively increases the expression of the gene encoding kinase suppressor of Ras-1 (KSR-1) in HL60 cells, while other differentiation-inducing agents such as 12-O-tetradecanoylphorbol-13-acetate, retinoic acid or dimethyl sulfoxide do not significantly increase KSR-1 expression. Further, the upregulation of KSR-1 gene by 1,25D is competed by ZK159222, an antagonist of vitamin D receptor (VDR) action, and can occur in the presence of protein synthesis inhibitor cycloheximide, showing that the effect is direct. Most importantly, we have identified a vitamin D responsive element (VDRE) in the promoter region of the human KSR-1 gene, to which VDR binds in a 1,25D-dependent manner, in vitro and in vivo. This binding is paralleled by increased association of RNA polymerase II with the transcription start site of KSR-1 gene, and the VDRE is functional in reporter assays. Our findings offer a potential mechanism for a signaling pathway that contributes to 1,25D-induced monocytic differentiation of human myeloid leukemia cells.