Indoleamine 2,3-dioxygenase (IDO) is an important enzyme in the regulation of immune responses; cells that express IDO can suppress T-cell responses and promote tolerance. Because of the critical role of endothelial cells in graft rejection, we have investigated the role of IDO expression by vascular endothelial cells and its consequence on immunoregulation. We compared the expression of IDO by primary human umbilical vein endothelial cells (HUVECs), human saphenous vein endothelial cells (HSVECs) and arterially derived endothelial cells using reverse transcriptase PCR, Western blotting and assays for enzymatic activity. In HUVECs IDO is upregulated by incubation with cytokines or in mycoplasma-infected cells. On the other hand HSVECs and arterially derived endothelial cells express little IDO, which is poorly upregulated upon activation (except by mycoplasma). Inhibition of IDO activity improved the ability of HUVECs to stimulate allogeneic T-cell responses. If either HUVECs or HSVECs are transfected with the gene encoding IDO, then they are incapable of stimulating allogeneic T-cell responses and induce anergy in allospecific T cells (which can also act as regulatory cells). The variable expression of IDO in different endothelial cells is important not only in understanding the role of endothelial cells in the regulation of graft rejection, but also as a potential therapeutic strategy.