T cell recognition of self antigens is a key event in the pathogenesis of autoimmune diseases. To date, the initial events that trigger autoreactive T cells are unknown. The "molecular mimicry" hypothesis predicts that during an infection T cells that recognize both a microbial antigen and a related self peptide become activated and cause autoimmune disease. We have systematically examined the recognition of self antigens by HLA-DR4-restricted T cells specific for peptides of the outer surface protein A (OspA) of Borrelia burgdorferi, the etiological agent of Lyme disease. We used the peptide spot synthesis technique for complete peptide substitution analyses of two immunodominant OspA epitopes. Each amino acid residue of the epitopes was substituted with all 20 naturally occurring amino acids and the altered peptides were tested for recognition by a panel of OspA-specific T cells. The binding motifs (supertopes) revealed by these analyses were used to screen public databases for matching human or murine peptides. Several hundred peptides were identified by this search and synthesized. Of these, 28 were recognized by OspA-specific T cells. Thus, T cell cross-reactivity is a common phenomenon and the existence of cross-reactive epitopes alone does not imply molecular mimicry-mediated pathology and autoimmunity.