It has been proposed that molecular mimicry between protein 2C (p2C) of coxsackie virus B4 and the autoantigen glutamic acid decarboxylase (GAD₆₅) plays a role in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). In this study we show that the amino acid sequence of p2C which shares homology with a sequence in GAD₆₅ (PEVKEK), is highly conserved in coxsackie virus B4 isolates as well as in different viruses of the subgroup of coxsackie B-like enteroviruses. These are the most prevalent enteroviruses and therefore exposure to the mimicry motif will be a frequent event throughout life. Presentation of the homologous peptides by HLA molecules is essential for T-cell reactivity. Therefore, we tested whether the PEVKEK motif can bind to the IDDM-associated HLA-DR1, -DR3 and -DR4 molecules. Synthetic peptides with sequences derived from p2C and GAD₆₅ did bind to HLA-DR3 but not to HLA-DR1 or -DR4. Replacement of amino acids within the motif showed that the PEVKEK motif binds specifically to HLA-DR3. Moreover, both p2C and GAD₆₅ peptides bind in the same position within the peptide binding groove of the DR3 molecule which is an essential requirement for T-cell cross-reactivity. The results support molecular mimicry between p2C of coxsackie B-like enteroviruses and GAD₆₅. However, this molecular mimicry may be limited to the HLA-DR3 positive subpopulation of IDDM patients. [Diabetologia (1998) 41: 40–46]