Dendritic cells (DCs) can induce tolerance or immunity. We identified and characterized an IDO-expressing and an IDO-negative human DC population after stimulation by various proinflammatory stimuli. IDO expression was strongly dependent on the maturation status of the cells (CD83-positive cells only). The two DC subpopulations remained IDO positive and IDO negative, respectively, over a time period of at least 48 h. IDO enzyme activity of human DCs was highest during stimulation by strongly maturation-inducing TLR ligands such as highly purified LPS (TLR4 ligand) or polyriboinosinic-polyribocytidilic acid (TLR3 ligand); factors of the adaptive immune system such as IFN-γ, a mixture of cytokines, and IFN-α had lesser stimulatory capacity for IDO induction and activity. After stimulation with CD40L, IDO-positive DCs expressed significantly increased levels of B7 family molecules such as CD40, CD80, CD86, ICOS ligand, as well as PD-L1 (B7-H1) and PD-L2 (B7-DC) compared with the IDO-negative DC subset. At the same time, the inhibitory receptors Ig-like transcripts 3 and 4 were significantly downregulated on IDO-positive cells. Functionally, IDO-positive DCs produced significantly more IL-1β and IL-15 and less IL-10 and IL-6 than the IDO-negative subset after CD40L stimulation. These results show that IDO expression is associated with a distinctive phenotype and functional capacity in mature DCs. It seems likely that the IDO-positive DC subset possesses a regulatory function and might skew a T cell response toward tolerance.