Direct injection of foreign antigen into the adult thymus is a potent route of antigen delivery for the induction of tolerance in vivo. In this report, we demonstrate that tolerance to C57BL/10 (H2b/BL10) alloantigens can be induced in CBA/Ca (H2k/CBA) mice by intrathymic (IT) administration of BL10 spleen leukocytes coincident with transient peripheral immunomodulation of CD4+ T cells using a depleting anti-CD4 monoclonal antibody. T cell receptor (TCR) transgenic mice (BM3.6; H2k) expressing a CD8-independent TCR specific for H2Kb were used as recipients to facilitate investigation of the mechanisms responsible for tolerance induction by allowing visualization of events in the thymus following IT injection. IT administration of 5 x 10(7) BL10 spleen leukocytes and concomitant transient peripheral T cell depletion in BM3.6 mice resulted in a substantial H2Kb-specific deletion of transgenic-TCR+ (tg-TCR) thymocytes which was dependent on the level of tg-TCR expression. IT deletion and the failure to export CD8+ T cells to the peripheral lymphoid organs correlated with the induction of tolerance to H2Kb; TCR transgenic mice that had received IT injection of BL10 splenocytes and peripheral T cell depletion accepted a H2Kb+ cardiac allograft indefinitely. Analysis of tolerant BM3.6 mice revealed that there were low numbers of CD8+ T cells in the periphery giving rise to a substantially reduced reactivity in vitro despite the fact that no donor cells or IT deletion were observed in the thymi of the majority of tolerant mice. These results demonstrate for the first time that IT injection of foreign alloantigen into an adult thymus results in the deletion of thymocytes expressing a TCR specific for the injected alloantigen and suggest that this is an important mechanism of tolerance induction following IT injection of alloantigen in vivo. Furthermore, analysis of tolerant TCR-transgenic mice suggests that IT deletion is not required for the maintenance of tolerance, and that peripheral mechanisms enforce continued hyporesponsiveness to H2Kb following transplantation.