Recent studies suggest that the adult immune system can be manipulated by intrathymic (IT) inoculation of donor Ag to accept cardiac and islet allografts in the low responder rat combination of Lewis-to-WF. We have now extended this study to examine the effect of IT inoculation of soluble protein Ag obtained from 3M KCl extracts of resting T cells combined with transient ALS immunosuppression on islet allograft survival in the high responder combination of WF-to-Lewis. We first confirmed the earlier observation that IT injection of 2 mg soluble Ag on day -7 led to permanent islet graft survival (>200 days) in the Lewis-to-WF rat combination without the use of recipient immunosuppression and found this to be true in the Lewis-to-ACI rat combination. In the high responder combination of WF-to-Lewis, unmodified Lewis rats pretreated with IT inoculation of 2 mg soluble Ag acutely rejected WF and BN islet allografts. IT inoculation of donor Ag combined with 1 ml ALS transient immunosuppression on day -7 led to a modest graft prolongation [24.8+/-10.1 days as compared with 15.2+/-3.6 days in ALS only treated controls]. Intrathymic injection of soluble Ag on day -7 combined with 1 ml ALS on days -7 and 0 relative to allografting resulted in 100% permanent islet graft survival (>200 days) compared with an MST of 20.6+/-2.3 days in ALS only-treated controls. Similar treatment led to acute rejection of 3rd party (BN) grafts, thus demonstrating donor-specificity. In addition, extrathymic inoculation of donor Ag in similarly immunosuppressed animals did not result in islet graft prolongation, once again confirming the importance of the thymus in tolerance induction. To examine them for donor-specific tolerance, long-term unresponsive (>120 days) Lewis recipients of renal subcapsular islets underwent nephrectomy of the islet bearing kidneys and were challenged with intraportal donor- or third party-type islets after becoming diabetic. All the nonimmunosuppressed recipients of donor-type (WF) islets became permanently normoglycemic (>100 days) while the third-party (BN) grafts were promptly rejected, with an MST of 10.6 days. These findings confirm that acquired thymic tolerance induced by IT inoculation of soluble protein Ag in the low to moderate responder rat combinations is reproducible in the high responder combination provided that adequate peritransplant immunosuppression is used. This study suggests that acquired thymic tolerance in the rat model is dependent on host responsiveness to alloantigens, MHC differences between the donor-recipient pair, and the use of transient immunosuppression in the high responder recipient. This model may have potential clinical application in the development of strategies for specific transplantation tolerance.