The active form of Vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], has potent antiproliferative actions on various normal and malignant cells. Calcemic effects, however, hamper therapeutic application of 1,25-(OH)(2)D(3) in hyperproliferative diseases. Two 14-epi-analogs of 1,25-(OH)(2)D(3) namely 19-nor-14-epi-23-yne-1,25-(OH)(2)D(3) (TX522) and 19-nor-14,20-bisepi-23-yne-1,25-(OH)(2)D(3) (TX527), display reduced calcemic effects coupled to an (at least 10-fold) increased antiproliferative potency when compared with 1,25-(OH)(2)D(3). Altered cofactor recruitment by the Vitamin D receptor (VDR) might underlie the superagonism of these 14-epi-analogs. Therefore, this study aims to evaluate their effects at the level of VDR-coactivator interactions. Mammalian two-hybrid assays with VDR and the coactivators TIF2 and DRIP205 showed the 14-epi-analogs to be more potent inducers of VDR-coactivator interactions than 1,25-(OH)(2)D(3). TX522 and TX527 require 30- and 40-fold lower doses to obtain the VDR-DRIP205 interaction induced by 1,25-(OH)(2)D(3) at 10(-8)M. Evaluation of additional 1,25-(OH)(2)D(3)-analogs and their impact on VDR-coactivator interactions revealed a strong correlation between the antiproliferative potency of an analog and its ability to induce VDR-coactivator interactions. In conclusion, these data show that altered coactivator binding by the VDR is one possible explanation for the superagonistic action of the two 14-epi-analogs TX522 and TX527.