The TCR alpha/beta chains recognize antigen peptides bound to the groove of the MHC class II molecule. The crystal structure analyses of the TCR/peptide/MHC class II complexes have revealed that the Valpha chains play a significant role in antigen recognition. However, molecular details which amino acid residues of the Valpha chain are able to contribute to fine antigen specificity are not clearly understood. Previously, we have classified a panel of T hybrids specific for insulin isotypes from different species of animals into four groups based on response profiles to these antigens. In particular, the group III (pork insulin > or = beef insulin hierarchy of responsiveness) and IV (pork insulin >> beef insulin hierarchy of responsiveness) T hybrids are interesting, since these TCR alpha/beta chains with marked different antigen specificities demonstrate identical gene usages and very similar sequences. To specifically address the molecular requirements for insulin recognition by TCR, the TCR alpha and beta chain genes from these group III and IV T hybrids were transfected into 58 alpha-beta- T hybrid. The experiments suggested that CDR3alpha dictates the fine antigen specificity. Then, we have introduced a series of mutations into position 95 of CDR3alpha. The mutation experiments clearly indicated that position 95alpha determines the antigen specificity of the group III and IV T hybrids.