Insulin-dependent diabetes mellitus (IDDM) results from selective autoimmune destruction of insulin producing beta-cells. T-cell reactivity and autoantibodies to several islet proteins such as insulin, GAD and IA-2 are associated with IDDM in mice and men. In NOD mice, the majority of T cells from insulitis specifically recognize the insulin B-chain peptide amino acid 9-22, in contrast to the periphery where the precursor frequency is much lower. It is important to note that these cells are diabetogenic. Surprisingly, the same insulin B-chain region contains epitopes recognized by protective T cells. In fact, autoimmune diabetes in NOD mice could be prevented by prophylactic treatment with this immunodominant T-cell epitope. In humans, however, no immunodominant regions of insulin have yet been defined. We have isolated and characterized a human insulin-specific T-cell clone that was derived from peripheral blood of a newly diagnosed IDDM patient. This patient displayed weakly positive primary T-cell responses to insulin. The peptide recognized by the clone was mapped to the insulin B chain (B:11-27). Functionally, the human insulin-specific CD4+ T cells displayed a Th1/0 like cytokine profile and were restricted by HLA-DR. The previously proposed alternative superantigen-like binding of insulin-B chain peptide outside of the peptide binding groove of HLA-DR could not be confirmed, since T-cell recognition was inhibited in competition experiments of insulin-B chain peptide with HLA-DR16 binding influenza peptide HA307-319. Our results indicate that human clonal T cells isolated from a recent onset IDDM patient recognize an epitope overlapping with the insulin B-chain region that is immunodominant and potentially therapeutic in NOD mice. This observation may be useful in studying the role of insulin-specific T cells in IDDM, and may eventually help to establish peptide-based immunotherapies in IDDM.