A single T cell clone can be activated by many different peptides in the context of a particular HLA molecule. To quantify the number of peptides that can be recognized by a CD4⁺ T cell clone, we screened a one‐bead‐one‐peptide synthetic peptide library and a protein database for peptides that stimulate an HLA‐DR3‐restricted, human glutamic acid decarboxylase (GAD65)‐reactive CD4⁺ T cell clone. Both the library screening and the database analysis indicated that this T cell clone is able to recognize approximately 10⁶ 11‐mer peptides at low nanomolar concentration. Furthermore, we determined that the frequency of cross‐reactivity increased only 1.5‐3 times when the peptide concentration increased 10 times, in the range of 0.01 – 1 μM. These data imply that there is a considerable potential for T cell cross‐reactivity and are useful for studies on the role of molecular mimicry in the etiology of T cell‐mediated disease.