Type 1 diabetes is thought to result from the destruction of β-cells by autoantigen-specific T-cells. Observations in the NOD mouse model suggest that CD8⁺ cytotoxic T-cells play an essential role in both the initial triggering of insulitis and its destructive phase. However, little is known about the epitopes derived from human β-cell autoantigens and presented by HLA class I molecules. We used a novel reverse immunology approach to identify HLA-A2−restricted, naturally processed epitopes derived from proinsulin, an autoantigen likely to play an important role in the pathogenesis of type 1 diabetes. Recombinant human proinsulin was digested with purified proteasome complexes to establish an inventory of potential COOH-terminals of HLA class I−presented epitopes. Cleavage data were then combined with epitope predictions based on the SYFPEITHI and BIMAS algorithms to select 10 candidate epitopes; 7 of these, including 3 with a sequence identical to murine proinsulin, were immunogenic in HLA-A2 transgenic mice. Moreover, six of six tested peptides were processed and presented by proinsulin-expressing cells. These results demonstrate the power of reverse immunology approaches. Moreover, the novel epitopes may be of significant interest in monitoring autoreactive T-cells in type 1 diabetes.