Type 1 diabetes results from the destruction of β-cells by an autoimmune T-cell response assisted by antigen-presenting B cells producing autoantibodies. CD8⁺ T-cell responses against islet cell antigens, thought to play a central role in diabetes pathogenesis, can be monitored using enzyme-linked immunosorbent spot (ELISpot) assays. However, such assays have been applied to monitoring of adult patients only, leaving aside the large and increasing pediatric patient population. The objective of this study was twofold: 1) to develop a CD8⁺ T-cell interferon-γ ELISpot assay for pediatric patients and 2) to determine whether zinc transporter 8 (ZnT8), a recently described target of autoantibodies in a majority of patients, is also recognized by autoreactive CD8⁺ T cells. Using DNA immunization of humanized mice, we identified nine HLA-A2–restricted ZnT8 epitopes. Among 36 HLA-A2⁺ children with diabetes, 29 responded to ZnT8 epitopes, whereas only 3 of 16 HLA-A2⁺ control patients and 0 of 17 HLA-A2⁻ control patients responded. Some single ZnT8 epitopes performed as well as the group of epitopes in discriminating between patients and control individuals. Thus, ZnT8 is a major CD8⁺ T-cell autoantigen, and ELISpot assays display similar performance in adult and pediatric type 1 diabetes.