Dendritic cells (DCs) play a central role in regulating immune activation and responses to self. DC maturation is central to the outcome of antigen presentation to T cells. Maturation of DCs is inhibited by physiological levels of 1alpha,25 dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] and a related analog, 1alpha,25(OH)(2)-16-ene-23-yne-26,27-hexafluoro-19-nor-vitamin D(3) (D(3) analog). Conditioning of bone marrow cultures with 10(-10) M D(3) analog resulted in accumulation of immature DCs with reduced IL-12 secretion and without induction of transforming growth factor beta1. These DCs retained an immature phenotype after withdrawal of D(3) analog and exhibited blunted responses to maturing stimuli (CD40 ligation, macrophage products, or lipopolysaccharide). Resistance to maturation depended on the presence of the 1alpha,25(OH)(2)D(3) receptor (VDR). In an in vivo model of DC-mediated antigen-specific sensitization, D(3) analog-conditioned DCs failed to sensitize and, instead, promoted prolonged survival of subsequent skin grafts expressing the same antigen. To investigate the physiologic significance of 1alpha,25(OH)(2)D(3)/VDR-mediated modulation of DC maturity we analyzed DC populations from mice lacking VDR. Compared with wild-type animals, VDR-deficient mice had hypertrophy of subcutaneous lymph nodes and an increase in mature DCs in lymph nodes but not spleen. We conclude that 1alpha,25(OH)(2)D(3)/VDR mediates physiologically relevant inhibition of DC maturity that is resistant to maturational stimuli and modulates antigen-specific immune responses in vivo.