The structural basis that determines the specificity of gammadelta T cell receptor (TCR) recognition remains undefined. Our previous data show that the complementary determining region of human TCRdelta (CDR3delta) is critical to ligand binding. Here we used linear and configurational approaches to examine the roles of V, N-D-N, or J regions in CDR3delta-mediated antigen recognition. Surprisingly, we found that the binding activities of CDR3delta from different gammadelta TCRs to their target tissues and ligands depend on the conserved flanking sequences (V and J) but not as much on the D region of CDR3delta fragment. We further defined the key residues in the V and J regions of CDR3delta fragments, including the cysteine residue in the V fragment and the leucine residue in the J fragment that determine their ligand binding specificity. Our results demonstrate that TCRdelta primarily uses conserved flanking regions to bind ligands. This finding may provide an explanation for the limited number of gammadelta TCR ligands that have as yet been identified.