Infections and TLR signals at the time of transplantation have been shown to prevent the induction of tolerance, but their effect on allografts after tolerance has been established is unclear. We here report that infection with Listeria monocytogenes precipitated the loss of tolerance and the MyD88- and T cell-dependent rejection of accepted cardiac allografts in mice. This loss of tolerance was associated with increases in the numbers of graft-infiltrating macrophages and dendritic cells, as well as CD4(+)FoxP3(-) and CD8(+) T cells. Rejection was also associated with increased numbers of graft-infiltrating alloreactive as well as Listeria-reactive IFNgamma-producing T cells. Rejection of the established grafts required both IL-6 and IFNss, cytokines produced during acute Listeria infection. However, IL-6 and IFNss alone, even when present at higher concentrations than during Listeria infection, were insufficient to break tolerance, while the combination of IL-6 and IFNss was sufficient to break tolerance. These and in vitro observations that IL-6 but not IFNss enhanced T cell proliferation while IFNss but not IL-6 enhanced IFNgamma production support a hypothesis that these cytokines play nonredundant roles. In conclusion, these studies demonstrate that the proinflammatory effects of infections can induce the loss of tolerance and acute rejection of accepted allografts.