Antigen-activated T lymphocytes undergo an immune or tolerogeneic response in part according to the activation status of their antigen-presenting cells. However, factors controlling the activation of antigen-presenting cells are not fully understood. In this study, we demonstrate that immune tolerance after organ allotransplantation in the rat is associated with a repressed intragraft expression of several enzymes of the trans-sulfuration pathway, including cystathionine γ-lyase (CSE). The pharmacologic blockade of CSE with propargylglycine delayed heart allograft rejection and abrogated type IV hypersensitivity but did not modify antibody responses, and was associated with a selective inhibition of the TH-1 type factors T-bet, IL-12, and IFN-γ. IL-12 repression could also be induced by propargylglycine in vitro in monocytes and dendritic cells (DCs), a phenomenon not mediated by changes to nuclear factor-κ B or hydrogen sulfide but that occurred together with a modulation of intracellular cysteine content. Intracellular cysteine levels were predominantly controlled in DCs by CSE activity, together with extracellular import via the X(c)(-) transporter. Our results indicate that CSE plays a critical role in regulating IL-12 in monocytes and DCs and is down-modulated in transplant tolerance, presumably participating in the maintenance of the tolerant state.