A utoimmune diabetes in nonobese diabetic (NOD) mice results from destruction of pancreatic β cells by T lymphocytes. It is believed that CD8⁺ cytotoxic T lymphocytes (CTLs) effect the initial β-cell insult in diabetes, but the mechanisms remain unclear. Studies of NOD.lpr mice have suggested that disease initiation is a Fas-dependent process, yet perforin-deficient NOD mice rarely develop diabetes despite expressing Fas. Here, we have investigated the role of perforin and Fas in the ability of β cell–reactive CD8⁺ T cells bearing a T-cell receptor (8.3-TCR) that is representative of TCRs used by CD8⁺ CTLs propagated from the earliest insulitic lesions of NOD mice, and that targets an immunodominant peptide/H-2K^d complex on β cells, to effect β-cell damage in vitro and in vivo. In vitro, 8.3-CTLs killed antigenic peptide–pulsed non–β-cell targets via both perforin and Fas, but they killed NOD β cells via Fas exclusively. Perforin-deficient 8.3-TCR–transgenic NOD mice expressing an oligoclonal or monoclonal T-cell repertoire developed diabetes even more frequently than their perforin-competent littermates. These results demonstrate that diabetogenic CD8⁺ CTLs representative of CTLs putatively involved in the initiation of autoimmune diabetes kill β cells in a Fas-dependent and perforin-independent manner.