We investigated the immune response to proinsulin, a potential autoantigen in IDDM secreted exclusively by pancreatic β-cells. A total of 2,142 short-term cell lines were generated from 19 individuals; seven IDDM patients at the disease onset and 12 control subjects. No increase in the frequency of proinsulin reactive cells was observed in the IDDM group. To define proinsulin epitopes, proliferative responses of proinsulin-specific lines were examined against 10 overlapping 15 amino acid peptides encompassing the human proinsulin sequence. The predominant immune response was directed against the proinsulin p35–50 peptide located in the (C) connecting peptide between the α- andβ-chain of insulin. Recognition of the proinsulin p35–50 peptide could be shown by generating specific T cell clones against the peptide. However, unlike responses to other tissue-specific autoantigens there were only low proliferative responses to proinsulin as measured by³H-thymidine incorporation. This low reactivity may be partially explained by the location of the p35–50 peptide in the C-peptide which is released into the circulation and therefore, may induce a clonal anergy of T reactive cells. However, the significantly higher³H-thymidine incorporation after CD3–CD28 triggering showed that peptide specific T cells were capable of a significant response with a stronger TCR signal.