Glutamic acid decarboxylase (GAD) 65 is a major autoantigen in type 1 diabetes. Regions of homology exist between GAD65 (residues 250-273) and the Coxsackie P2-C protein (residues 28-50) and between GAD65 (residues 506-518) and proinsulin (residues 24-36), and each of these has been reported to be a diabetes-associated T cell target. The aim of this study was to determine whether the homologous regions are shared targets of T lymphocyte reactivity in individual patients with type 1 diabetes. T cell proliferation against the corresponding peptide pairs, GAD254-276 and Coxsackie P2-C32-54 and GAD506-518 and proinsulin24-36, were measured in peripheral blood mononuclear cells from 26 patients with newly diagnosed type 1 diabetes and 24 control subjects. Responses with stimulation indices higher than 3 were found against each of the antigens tested in both patients and control subjects, and no differences were observed between groups. A strong positive correlation was found between responses to the corresponding peptide pairs GAD254-276 and Coxsackie P2-C32-54 (r=0.77, P<0.0001), and between responses to the corresponding peptide pairs GAD506-518 and proinsulin24-36 (r=0.66, P<0.0001). However, a similar correlation was also observed between responses to the noncorresponding pairs Coxsackie P2-C32-54 and proinsulin24-36 (r=0.82, P<0.0001), Coxsackie P2-C32-54 and GAD506-518 (r=0.82, P<0.0001), and GAD254-276 and proinsulin24-36 (r=0.83, P<0.0001). Strikingly, increased responses to peptides were found almost exclusively in subjects with high stimulation indices against the recall antigen tetanus toxoid, further suggesting that peripheral blood T cell responses are related to a general subject hyperreactivity. These data suggest that proliferative T cell responses to peptides containing putative autoreactive epitopes of GAD65 and proinsulin are not specific for type 1 diabetes, that correlation between T cell reactivity to peptides is not restricted to those containing homologous regions, and that non-antigen-specific factors are important determinants of in vitro measurements of T cell reactivity.