Induction of tolerance to fully major histocompatibility complex (MHC)-mismatched rat islet allografts implanted at two different islet transplant sites (liver and kidney capsule [KC]) was examined. Streptozotocin-induced diabetic Lewis (RT1(1)) rats remained hyperglycemic (> 200 mg/dl) after intrahepatic preimmunization by injection of 200 low-temperature cultured (24 degrees C for 7 days) Wistar-Furth (WF, RT1u) rat islets into the portal vein with one injection (1 ml) of rat antilymphocyte serum intraperitoneally. Three weeks later, 1,200 WF islets that had been cultured to remove passenger lymphoid cells were transplanted into the liver via the portal vein or under the KC. The intrahepatic transplants survived 60.2 +/- 11.9 days, and all six of the KC transplants maintained normoglycemia for > 100 days after the preimmunization regimen. In contrast, survival of fresh islet transplants was not significantly improved by this preimmunization protocol at either transplantation site. This study demonstrates that indefinite islet allograft survival can be achieved across a full MHC mismatch by intrahepatic preimmunization with a small number of cultured donor islets and a brief period of immunosuppression followed by transplantation of low-temperature cultured donor islets.