To study the effects of the active metabolite of vitamin D(3), 1,25(OH)(2)D(3), an immunomodulatory hormone, on the generation of so-called immature dendritic cells (iDCs) generated from monocytes (Mo-iDCs). Human peripheral blood monocytes were cultured to iDCs in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 for 1 week, with or without the extra addition of 10(-8) M 1,25(OH)(2)D(3) to the culture. Their phenotypes (CD14, CD1a, CD83, HLA-DR, CD80, CD86 and CD40 expression) were examined by fluorescence-activated cell sorting, and their T-cell stimulatory potential was investigated in allogeneic mixed lymphocyte reaction (allo-MLR). Additionally, their in vitro production of IL-10, IL-12 and transforming growth factor beta (TGF-beta) were examined by using the enzyme-linked immunosorbent assay. When 1,25(OH)(2)D(3) was added to monocytes in culture with GM-CSF and IL-4, it hampered the maturation of Mo-iDCs. First, the phenotype of the 1,25(OH)(2)D(3)-differentiated DCs was affected, there being impaired downregulation of the monocytic marker CD14 and impaired upregulation of the markers CD1a, CD83, HLA-DR, CD80 and CD40. CD86 was expressed on more 1,25(OH)(2)D(3)-differentiated DCs. Secondly, the T-cell stimulatory capability of 1,25(OH)(2)D(3)-differentiated DCs was upregulated relative to the original monocytes to a lesser degree than DCs differentiated without 1,25(OH)(2)D(3) when tested in an allo-MLR. With regard to the production of cytokines, Staphylococcus aureus cowan 1 strain (SAC)-induced IL-10 production, although not enhanced, remained high in 1,25(OH)(2)D(3)-differentiated DCs, but was strongly downregulated in DCs generated in the absence of 1,25(OH)(2)D(3). SAC/interferon-gamma-induced IL-12 production was clearly upregulated in both types of DC relative to those of the original monocytes, and TGF-beta production was downregulated. Our data confirm earlier reports showing that 1,25(OH)(2)D(3) hampers the maturation of fully active immunostimulatory major histocompatibility complex (MHC) class II+, CD1a+, CD80+ DCs from monocytes. Our data supplement the data from other reports by showing that the expression of CD86 was upregulated in 1,25(OH)(2)D(3)-differentiated DCs, whilst the capacity for IL-10 production remained high. Collectively, these data are in line with earlier descriptions of suppressive activities of this steroid-like hormone with respect to the stimulation of cell-mediated immunity.