NOD mouse-derived β-cell-specific cytotoxic T-cell (β-CTL) clones are diabetogenic in adult NOD mice, but only if co-injected with splenic CD4⁺ T-cells from diabetic animals. This investigation was initiated to determine whether infiltration of pancreatic islets by β-CTL is a major histocompatibility complex (MHC) class I-restricted response, and whether β-CTL has a direct cytopathic effect on β-cells in vivo. Pancreatic islets from BALB/c (H-2^d) or B6 (H-2^b) mice were transplanted under the renal capsule of streptozotocin (STZ)-induced diabetic (NOD × BALB/c) Fl (H-2K^d, H-2D^d,b) or (NOD × B6) Fl (H-2K^d,b, H-2D^b) mice, respectively. H-2K^d-restricted β-CTL clones from NOD mice were transfused into euglycemic mice within 3 days after transplantation. In all of the H-2^d islet-grafted (NOD × BALB/c) Fl mice that received the β-CTL clones, the β-CTLs homed into the grafts, recruited host Mac-1⁺ cells and CD4⁺ and CD8⁺ T-cells, and caused diabetes within 7 days. In contrast, none of the H-2^b islet-grafted (NOD × B6) Fl mice who received the β-CTL clones and none of the H-2^d islet-grafted (NOD × BALB/c) Fl mice who received a non-β-cell cytotoxic CTL clone (Nβ-CTL) developed graft inflammation or diabetes. Depletion of CD4⁺ T-cells in H-2^d islet-grafted (NOD × BALB/c) Fl mice did not prevent β-CTL clone-induced diabetes but reduced its severity. In contrast, when the β-CTL clones were injected >8 days after transplantation, none of the H-2^d islet-grafted (NOD × BALB/c) Fl mice became diabetic or developed graft inflammation. We conclude that (1) isletderived β-CTLs can destroy β-cells in vivo; (2) infiltration of grafted islets by β-CTLs is an MHC class I-restricted response; (3) β-CTLs can recruit naive CD4⁺ T-cells to the site, leading to further β-cell damage; and (4) revascularized islet grafts are, like pancreatic islets of irradiated adult NOD mice, “sequestered” from circulating β-CTLs.