1α, 25-Dihydroxyvitamin D 3 (1, 25 (OH) 2 D 3), the active form of vitamin D 3, is a potent immunomodulatory agent. Here we show that dendritic cells (DCs) are major targets of 1, 25 (OH) 2 D 3-induced immunosuppressive activity. 1, 25 (OH) 2 D 3 prevents the differentiation in immature DCs of human monocytes cultured with GM-CSF and IL-4. Addition of 1, 25 (OH) 2 D 3 during LPS-induced maturation maintains the immature DC phenotype characterized by high mannose receptor and low CD83 expression and markedly inhibits up-regulation of the costimulatory molecules CD40, CD80, and CD86 and of class II MHC molecules. This is associated with a reduced capacity of DCs to activate alloreactive T cells, as determined by decreased proliferation and IFN-γ secretion in mixed leukocyte cultures. 1, 25 (OH) 2 D 3 also affects maturing DCs, leading to inhibition of IL-12p75 and enhanced IL-10 secretion upon activation by CD40 ligation. In addition, 1, 25 (OH) 2 D 3 promotes the spontaneous apoptosis of mature DCs. The modulation of phenotype and function of DCs matured in the presence of 1, 25 (OH) 2 D 3 induces cocultured alloreactive CD4+ cells to secrete less IFN-γ upon restimulation, up-regulate CD152, and down-regulate CD154 molecules. The inhibition of DC differentiation and maturation as well as modulation of their activation and survival leading to T cell hyporesponsiveness may explain the immunosuppressive activity of 1, 25 (OH) 2 D 3.