Autoimmune (type 1) diabetes mellitus results from the destruction of insulin-producing pancreatic beta-cells by T lymphocytes. Beta-cell death that is induced by autoreactive CTL in diabetes involves both Fas/Fas ligand (FasL)- and perforin/granzyme-mediated pathways, although their relative contributions during the progression of the disease remain unknown. We demonstrate here that despite the preferential use of the Fas/FasL pathway for cytolysis of beta-cell targets, transgenic beta-cell-specific CTL were able to kill targets via the perforin pathway when triggered by a higher affinity stimulus. In addition, we show that the killing mechanism used by islet-associated CD8(+) T cells from non-obese diabetic mice changed as the mice aged and correspondingly, with the stage of diabetes. These results provide direct evidence for age-related changes in the cytotoxic pathways used by diabetogenic T cells during the progression of autoimmune diabetes.