Aims: Cells that express indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in the catabolism of tryptophan, suppress T-cell responses and promote immunological tolerance. However, their role in solid organ transplantation is incompletely understood. We analyzed T-cell responses to allogeneic dendritic cells (DCs) genetically modified to express the gene encoding IDO in vitro, and IDO gene transfer into the donor heart in a cardiac transplant model in vivo. Methods and Results: Bone marrow-derived DCs transduced with the gene encoding IDO produced active IDO protein. This was associated with decreased stimulation of allogeneic T-cell proliferation in the mixed leukocyte reaction in vitro. In a cardiac transplant model, adenovirus-mediated IDO gene transfer into the donor heart resulted in transgene expression predominantly in cardiomyocytes. F344 rat donor hearts transduced with the gene encoding IDO survived for longer periods of time when placed in Lewis recipients compared with control vector or vehicle alone (median survival time, 17 [range: 12-22] days vs. 10 [8-14] and 9 [8-13] days, respectively; p<0.0001). IDO gene transfer combined with low-dose cyclosporin A (CsA) was more effective than CsA alone (p<0.05). Numbers of monocytes/macrophages, CD4⁺ cells, and CD8⁺ cells infiltrating the graft, and intragraft cytokine transcript levels for IFN-, IL-1, TNF-, TGF- and RANTES/CCL5 were decreased after IDO gene transfer (p<0.05). Conclusions: DCs genetically engineered to overexpress IDO modulate T-cell alloresponses in vitro. IDO gene transfer into the donor heart attenuates acute cardiac allograft rejection. Regulation of tryptophan catabolism by means of IDO overexpression may be a useful approach in heart transplantation.