GAD65 contains an amino acid sequence which is highly homologous with a sequence in the 2C protein of coxsackievirus B4 (CBV4-2C). In the present study the possibility that this region could contain an epitope capable of inducing immunological cross-reactivity between CBV4-2C and GAD65 was evaluated. Six out of seven rabbit sera, which were raised against seven different synthetic peptides carrying various modifications of the homology sequence, showed cross-reactivity between 2C. GAD65 and GADD67 derived peptides in ELISA. There was substantial cross-reactivity between 2C and GAD65 peptides, but not between 2C and GAD67 peptides. The most cross-reactive peptides were those corresponding to the 2C sequences FIEWLKVKILPEVKEK and KILPEVKEKHEFLSRL. When the binding of the four 2C peptide-specific sera to the GAD65 protein was analysed in immunoprecipitation, two sera were found to be cross-reactive (anti-FIEWLKVKILPEVKEK and anti-WLKVKILPEVKEKHEF). One of these (anti-WLKVKILPEVKEKHEF) reacted also with coxsackie B virus (CBV)-infected cells. Antibodies against this epitope were induced during enterovirus (including CBV) infections in initially healthy children who later progressed to clinical insulin-dependent diabetes mellitus (IDDM). Antibody responses were frequent also in constantly GAD65 antibody-negative non-diabetic children, and antibody levels did not differ between newly diagnosed IDDM patients and matched control subjects. Blocking experiments confirmed that the observed reactivity of both rabbit and human antibodies was immunologically specific. The results suggest that the epitope is antigenically highly similar in 2C and GAD65, and that peptide immunization induces antibodies which cross-react with these molecules. However, the significance of this phenomenon in the pathogenesis of IDDM remains to be confirmed, as the peptide antibody levels were similar in patients with recent-onset IDDM and in control subjects.