1,25-Dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) plays a major role in the stimulation of bone growth, mineralization, and intestinal calcium and phosphate absorption; it also acts as a general inhibitor of cellular proliferation. Several new, clinically relevant compounds dissociate antiproliferative and calcemic activities of 1,25(OH) 2 D 3 , but the molecular basis for this has not been clearly elucidated. Here, we tested whether the potency of one class of compounds, 20-epi analogues, to induce myeloid cell differentiation, is because of direct molecular effects on vitamin D receptor (VDR). We report that two 20-epi analogues, MC1627 and MC1288, induced differentiation and transcription of p21Waf1,Cip1 , a key VDR target gene involved in growth inhibition, at a concentration 100-fold lower than that of 1,25(OH) 2 D 3 . We compared this sensitivity to analogue effects on VDR interacting proteins: RXR, GRIP-1, and DRIP205, a subunit of the DRIP coactivator complex. Compared with the interaction of VDR with RXR or GRIP-1, the differentiation dose-response most closely correlated to the ligand-dependent recruitment of the DRIP coactivator complex to VDR and to the ability of the receptor to activate transcription in a cell-free system. These results provide compelling links between the efficiency of the 20-epi analogue in inducing VDR/DRIP interactions, transactivation in vitro, and its enhanced ability to induce cellular differentiation.In the early part of this century, vitamin D 3 was discovered as a fat-soluble substance with antirachitic activity (1). It was subsequently found that vitamin D 3 must be metabolized to an active hormonal form before it can elicit its biological effects (2). Upon exposure to UV light, vitamin D 3 is synthesized, converted in the liver to 25-hydroxyvitamin D 3 , and hydroxylated in the kidney to its active form, 1,25-dihydroxyvitamin D3 (1,25(OH) 2 D 3 ).1 Functions of 1,25(OH) 2 D 3 include the stimulation of bone growth and mineralization and the increase intestinal calcium and phosphate absorption. In recent years, accumulating evidence points to the involvement of 1,25(OH) 2 D 3 in a number of diverse biological processes, including the regulation of cellular proliferation and differentiation, immunosuppression, and hormone secretion.Similar to other steroid hormones, 1,25(OH) 2 D 3 acts by binding stereospecifically to a high affinity, low capacity intracellular receptor protein, the vitamin D receptor (VDR) (3). VDR is a 48-kDa protein and is a member of the steroid/nuclear receptor superfamily that contains discrete functional domains for ligand binding, DNA binding, and transcriptional activation (4). Transactivation by 1,25(OH) 2 D 3 requires a carboxyl-terminal ␣-helical region, termed activation function-2 (AF-2), that forms part of the ligand-binding pocket (5). Upon binding to 1,25(OH) 2 D 3 , VDR dimerizes with the retinoid X receptor (RXR) to enable high affinity interactions with a specific vitamin D-responsive element (VDRE) located in 5Ј-flanking regions of target g...