The hormonal active form of vitamin D3, 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3), inhibits (through an unknown mechanism) the ability of monocytes/macrophages to induce T‐cell activation. For T cells to be optimally activated, recognition of antigen/major histocompatibility complexes (MHC) by the T‐cell receptor (TCR) must be accompanied by a second costimulatory signal. Considerable experimental data now suggest that this costimulatory signal is predominantly generated by B7.1 and/or B7.2 molecules, expressed on antigen‐presenting cells (APC), when engaged to their counter‐receptor, CD28, present on T cells. To determine whether the inhibitory effect of 1,25(OH)2D3 on monocytes/macrophages might involve modulation of the expression of B7.1 and B7.2 molecules, we analysed (by flow cytometry) the influence of 1,25(OH)2D3 and an analogue, KH 1060, on the expression of these two molecules at the surface of resting human peripheral blood monocytes. In parallel, we tested the effect of these two agents on human monocyte expression of cell‐surface markers (CD14 and CD4) and antigen‐presenting molecules (MHC class I and MHC class II). Our results showed that both 1,25(OH)2D3 and KH 1060 inhibited the basal expression of B7.2 in a dose‐ and time‐dependent manner, without affecting B7.1. Moreover, these two compounds increased CD14 and reduced MHC class II and CD4 expression. Furthermore, the effect of 1,25(OH)2D3 on B7 molecule expression in combination with lipopolysaccharide (LPS) or cytokines, including interleukin‐10 (IL‐10), interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α), was studied. The 1,25(OH)2D3‐induced B7.2 down‐regulation was still detectable when monocytes were activated by IL‐10, IFN‐γ and TNF‐α but not with LPS. Moreover, the induction of B7.1 by TNF‐α was inhibited by addition of 1,25(OH)2D3. We conclude that the ability of 1,25(OH)2D3 to decrease B7.2 expression on human monocytes might contribute to its inhibitory effect on APC‐dependent T‐cell activation and to its immunosuppressive properties observed in autoimmune diseases and organ transplantation.