1,25-Dihydroxyvitamin D3 (D3) exerts its effects by binding to and activating nuclear vitamin D3 receptors (VDRs) that regulate transcription of target genes. We have investigated regulation of VDR levels in human skin in vivo and in cultured human keratinocytes. Quantitative ligand-binding analysis revealed that human skin expressed approximately 220 VDRs per cell, which bound D3 with high affinity [(dissociation constant (Kd) = 0.22 nM]. In human skin nuclear extracts, VDR exclusively bound to DNA containing vitamin D3 response elements as heterodimers with retinoid X receptors. Topical application of D3 to human skin elevated VDR protein levels 2-fold, as measured by both ligand-binding and DNA-binding assays. In contrast, the D3 analog calcipotriene had no effect on VDR levels. Topical D3 had no effect on VDR mRNA, indicating that D3 either stimulated synthesis and/or inhibited degradation of VDRs. To investigate this latter possibility, recombinant VDRs were incubated with skin lysates in the presence or absence of D3. The presence of D3 substantially protected VDRs against degradation by human skin lysates. VDR degradation was inhibited by proteasome inhibitors, but not lysosome or serine protease inhibitors. In cultured keratinocytes, D3 or proteasome inhibitors increased VDR protein without affecting VDR mRNA levels. In cells, VDR was ubiquitinated and this ubiquitination was inhibited by D3. Proteasome inhibitors in combination with D3 enhanced VDR-mediated gene expression, as measured by induction of vitamin D3 24-hydroxylase mRNA in cultured keratinocytes. Taken together, our findings indicate that low VDR levels are maintained, in part, through ubiquitin/proteasome-mediated degradation and that low VDR levels limit D3 signaling. D3 exerts dual positive influences on its nuclear receptor, simultaneously stimulating VDR transactivation activity and retarding VDR degradation.