Cross-reactivity between an autoantigen and unknown microbial epitopes has been proposed as a molecular mechanism involved in the development of insulin-dependent diabetes (type 1 diabetes). Type 1 diabetes is an autoimmune disease that occurs in humans and the nonobese diabetic (NOD) mouse. BDC2.5 is an islet-specific CD4+ T-cell clone derived from the NOD mouse whose natural target antigen is unknown. A biometrical analysis of screening data from BDC2.5 T-cells and a positional scanning synthetic combinatorial library (PS-SCL) was used to analyze and rank all peptides in public viral and bacterial protein databases and identify potential molecular mimic sequences with predicted reactivity. Selected sequences were synthesized and tested for stimulatory activity with BDC2.5 T-cells. Active peptides were identified, and some of them were also able to stimulate spontaneously activated T-cells derived from young, pre-diabetic NOD mice, indicating that the reactivity of the BDC2.5 T-cell is directed at numerous mouse peptides. Our results provide evidence for their possible role as T-cell ligands involved in the activation of diabetogenic T-cells.