CD8+ islet cell-specific CTL lines and clones were established from lymphocytes infiltrating the pancreatic islets of acutely diabetic nonobese diabetic (NOD) mice from two subcolonies (NOD/Yn and NOD/Lt). CTL from NOD/Yn mice were predominantly cytotoxic against H-2b+ islet cells and to a lesser extent against H-2d+ islet cells. On the other hand, CTL from NOD/Lt mice were cytotoxic against H-2d+ but not against H-2b+ islet cells. Three of four CTL clones derived from NOD/Yn mice were H-2Db restricted, whereas two of two CTL clones derived from NOD/Lt mice were H-2Kd restricted. However, all of the H-2Kd restricted T cell clones expressed the same TCR, regardless of the NOD subcolony from which they were derived, compatible with a restricted repertoire. When two representative CTL clones were transferred into irradiated young NOD mice, neither induced insulitis or diabetes. However, transfer of these clones, together with CD4(+)-rich NOD splenocytes depleted of CD8+ T cells, caused severe insulitis and diabetes. When recipient NOD mice were treated with anti-CD4 mAbs, none of the mice developed insulitis or diabetes. Most of the irradiated NOD mice that received CD8-depleted splenocytes alone did not become diabetic. Through these studies we show that CTL clones can destroy pancreatic beta-cells as final effectors but that these clones require signals from CD4+ T cells to effect beta-cell damage.