Human peripheral gamma delta T lymphocytes are characterized by the preferential expression of a TCR consisting of a V delta 2-J delta 1-C delta chain and a V gamma 9-J gamma 1.2-C gamma 1 chain, which are virtually absent on thymocytes. Here we report the identification of a unique selection determinant that is located in the polyclonal V delta 2-J delta 1 junctional regions of peripheral gamma delta T lymphocytes. The selection determinant was discovered by the presence of an invariant T nucleotide at the relative second position of the V delta 2-J delta 1 junctional regions of peripheral polyclonal gamma delta T lymphocytes. Comparison of published sequences from peripheral gamma delta T lymphocytes confirmed the presence of this invariant T nucleotide (90%) in healthy individuals and in patients with various diseases. Translation of the relative first codon of the V delta 2-J delta 1 junctional regions revealed strikingly high frequencies of the homologous hydrophobic amino acids leucine (46%), valine (35%), and isoleucine (5%) at this position. The invariant T nucleotide was absent in polyclonal thymocytes and out-of-frame V delta 2-J delta 1 junctional regions, which proves that selection occurred at the protein level and not at the genomic level. No selection determinant could be identified in V gamma 9-J gamma 1.2 junctional regions, but the frequently occurring invariable, so-called canonical junctional region provided evidence for biased recombination processes. Although the obtained data do not allow discrimination between thymic selection and/or peripheral Ag-driven expansion, the identification of a strong selection determinant consisting of only one amino acid at a fixed position in V delta 2-J delta 1 junctional regions of virtually all peripheral polyclonal V delta 2/V gamma 9 T lymphocytes provides a new perception of TCR specificity and selection processes at the TCR protein level.