The active form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), exerts its effects through regulation of target gene transcription. Configuration at C-20 of 1,25(OH)(2)D(3) is important in determining potency, as shown by the high potency of analogs with inverted configuration at C-20 (20-epi compounds). Gemini analogs of 1,25(OH)(2)D(3) contain two side chains, combining a C-20-normal with a C20-epi side chain. We studied the potency of analogs combining double (Gemini) side chains with a 23-triple bond and a C-26,27-hexafluoro substitution in either the 20-epi (analog 20R) or 20-normal (analog 20S) side chain. These novel Gemini analogs were 8-50-fold more potent than 1,25(OH)(2)D(3) in inducing U937, HL-60G, and THP-1 differentiation and 5-50-fold more potent in inducing transcription from the osteocalcin vitamin D response element or the 25-hydroxyvitamin D(3)-24-hydroxylase (24OHase) promoter. In vivo, following i.p. injection in vitamin D-deficient mice, the 20S analog induced significantly higher levels of calbindin-D(9K) mRNA in intestine, and 24OHase and calbindin-D(28K) in kidney than 1,25(OH)(2)D(3) or analog 20R. Increased potency did not correlate with ligand-receptor binding affinity. In GST-pull down assays using in vitro translated VDR, Gemini analogs showed equivalent (or even attenuated) potency to 1,25(OH)(2)D(3) in recruiting cofactors DRIP205 and GRIP-1 to VDR. However, Gemini analogs were up to 15-fold more potent than 1,25(OH)(2)D(3) in recruiting the same cofactors to VDR in GST-pull down assays using equal amounts of VDR from nuclear extracts of VDR transfected and hormone treated (24 hr) COS-7 cells. Deletion of C-19 in either 20S or 20R Gemini analogs resulted overall in slightly less potent analogs compared to Gemini itself. We conclude that enhanced potency of the novel Gemini analogs is at least partly due to increased metabolic stability of the analogs, resulting in more cofactor binding and elevated levels of transcription.