1,25-Dihydroxyvitamin D3 (VD) controls multiple aspects of homeostasis, cell growth, and differentiation by the action of its nuclear receptor (VDR), which binds to, and activates transcription from, response elements in the promoter region of its target genes. Carbonic anhydrase-II (CA-II), an enzyme important to osteoclast function, has been shown to be regulated by VD. We screened the promoter of chicken CA-II for VDR binding sites and identified a functional VDRE, between positions -1,203 and -1,187. Like the majority of the VDREs described to date, this response element consists of two directly repeated hexameric core binding motifs spaced by three nucleotides and is bound by a heterodimer formed by the VDR and the retinoid X receptor (RXR). We show that the polarity of the binding of this heterodimer is 5'-VDR-RXR-3' in the CA-II VDRE, whereas on a "classical" DR3-type VDRE, such as that of the mouse osteopontin gene, this polarity is reversed to 5'-RXR-VDR-3'. We also show that the polarity of the heterodimeric complex in relation to the basic transcriptional machinery influences the sensitivity of the transcriptional activity to VD. This suggests that the orientation of a hormone response element in its natural promoter context constitutes an additional level of gene regulation.