Activation of autoreactive T cells is a crucial event in the pathogenesis of autoimmune diseases. Cross-reactivity between microbial and self Ags (molecular mimicry) is one hypothesis that could explain the activation of autoreactive T cells. We have systematically examined this hypothesis in experimental autoimmune encephalomyelitis using mice bearing exclusively myelin basic protein (MBP)-specific T cells (designated T+ α−). A peptide substitution analysis was performed in which each residue of the MBP Ac1–11 peptide was exchanged by all 20 naturally occurring amino acids. This allowed the definition of the motif (supertope) that is recognized by the MBP Ac1–11-specific T cells. The supertope was used to screen protein databases (SwissProt and TREMBL). By the search, 832 peptides of microbial origin were identified and synthesized. Of these, 61 peptides induced proliferation of the MBP Ac1–11-specific transgenic T cells in vitro. Thus, the definition of a supertope by global amino acid substitution can identify multiple microbial mimic peptides that activate an encephalitogenic TCR. Peptides with only two native MBP-residues were sufficient to activate MBP Ac1–11-specific T cells in vitro, and experimental autoimmune encephalomyelitis could be induced by immunizing mice with a mimic peptide with only four native MBP residues.