Type 1 diabetes (T1D) is caused by T cell-mediated destruction of the pancreatic insulin-producing β cells. While the role of CD4+ T cells in the pathogenesis of T1D is accepted widely, the epitopes recognized by pathogenic human CD4+ T cells remain poorly defined. None the less, responses to the N-terminal region of the insulin A-chain have been described. Human CD4+ T cells from the pancreatic lymph nodes of subjects with T1D respond to the first 15 amino acids of the insulin A-chain. We identified a human leucocyte antigen-DR4-restricted epitope comprising the first 13 amino acids of the insulin A-chain (A1-13), dependent upon generation of a vicinal disulphide bond between adjacent cysteines (A6–A7). Here we describe the analysis of a CD4+ T cell clone, isolated from a subject with T1D, which recognizes a new HLR-DR4-restricted epitope (KRGIVEQCCTSICS) that overlaps the insulin A1-13 epitope. This is a novel epitope, because the clone responds to proinsulin but not to insulin, T cell recognition requires the last two residues of the C-peptide (Lys, Arg) and recognition does not depend upon a vicinal disulphide bond between the A6 and A7 cysteines. The finding of a further CD4+ T cell epitope in the N-terminal A-chain region of human insulin underscores the importance of this region as a target of CD4+ T cell responses in human T1D.