The nuclear hormone 1a,25-dihydroxyvitamin D 3 (1a,25(OH) 2 D 3 ) acts through the transcription factor vitamin D receptor (VDR) via combined contact with the retinoid X receptor (RXR), coactivator proteins, and speci®c DNA binding sites (VDREs). Ligand-mediated conformational changes of the VDR are the core of the molecular switch of nuclear 1a,25(OH) 2 D 3 signalling. Studying the interaction of 1a,25(OH) 2 D 3 analogues with this molecular switch should allow the characterization of their potential selective biological pro®le. A 1a,25(OH) 2 D 3 analogue with two side chains (Ro27-2310 or Gemini) was found to stabilize functional VDR conformations and VDR±RXR heterodimers on a VDRE with a slightly lower sensitivity than the natural hormone. A 19-nor derivative of Gemini (Ro27-5646) showed similar sensitivity whereas 5,6-trans (Ro27-6462) 3-epi (Ro27-5840) and 1a-¯uoro (Ro27-3752) derivatives were equal to each other, but approximately 30-times less sensitive than Gemini. A des-C,D derivative of Gemini (Ro28-1909) showed only residual activity at maximal concentrations. In contrast to 1a,25(OH) 2 D 3 , Gemini and its derivatives showed a differential preference in stabilizing VDR conformations which was found to be modulated by DNA coactivator and corepressor proteins. An analysis of the gene regulatory potential of the VDR agonists in cellular reporter gene systems demonstrated the same ranking as in the in vitro systems, but Gemini and its 19-nor derivative were found to be more sensitive than 1a,25(OH) 2 D 3 which indicates that the natural hormone is selectively metabolized. This study used straightforward methods for the in vitro and ex vivo evaluation of the gene regulatory potential of 1a,25(OH) 2 D 3 analogues. Gemini was highlighted as an interesting drug candidate which could not be optimized through obvious chemical modi®cations in its A-ring.