Intrathymic (i.t.) injection of donor alloantigens has proved to be an effective strategy for the induction of tolerance. However, the mechanisms by which tolerance is induced and maintained after transplantation remain unclear. In this report we show that tolerance to donor cardiac allografts can be induced across a MHC class I difference by i.t. injection of donor splenocytes and transient T cell depletion. Furthermore, using H-2K(b)-specific TCR transgenic mice (BM3), we demonstrate that prolonged deletion of donor-reactive thymocytes was essential to induce tolerance by i.t. injection and this was dependent upon donor cells persisting in the thymus. Examination of the kinetics of thymic export following i.t. injection revealed that prolonged deletion of thymocytes was required to delay export of new T cells to the periphery until the time of transplantation. Importantly, after transplantation donor cell persistence in the thymus and i.t. deletion were no longer necessary to maintain tolerance. The graft itself or cells from the graft was responsible for maintaining tolerance at this stage. These findings reveal that multiple mechanisms are responsible for the induction and maintenance phases of tolerance to alloantigens in vivo after i.t. delivery, and that a complex inter-relationship between donor cell persistence in the thymus, i.t. deletion, thymic export of T cells and the timing of transplantation is involved.