We have shown that features of infectious tolerance, as originally described in thymectomized mice, may be applied to euthymic rat recipients of heart transplants. We now report on studies aimed at exposing mechanisms underlying the infectious tolerance pathway, with emphasis on the role of thymus and alloantigen. Pretransplant thymectomy diminished the efficacy of CD4-targeted therapy, with donor-specific tolerance induced in∼ 50% of recipients. Thymus was required for generation of regulatory T cells under the cover of CD4 mAb therapy and for the ability of these cells to confer infectious tolerance. However, thymus was not mandatory to maintain an infectious-permissive environment in cohorts of adoptively transferred recipients. Intragraft expression of IL-2, IL-4, and IL-10 genes was diminished in euthymic and thymectomized tolerant hosts. However, grafts in the latter group showed significant IFN-γ gene expression, suggesting a less efficient down-regulation of Th1-like cells in the absence of regulatory cells. Indeed, exogenous challenge with rIL-2 or freshly alloactivated spleen cells recreated rejection in thymectomized, but not euthymic, hosts, suggesting that a state of cytokine-responsive anergy contributes to the “noninfectious” form of tolerance in thymectomized rats. The infection-tolerant state did not result from “graft adaptation,” and regulatory T cells restricted for the original alloantigen were exposed to its continuous stimulation. The effective memory for suppression was dependent upon persistent donor-specific alloantigen stimulation; it disappeared within 3 weeks after its removal. Hence, both central and peripheral immune mechanisms, orchestrated by the tolerizing alloantigen, contribute to the infectious tolerance pathway in CD4 mAb-treated rat transplant recipients.