Careful analysis of functional V delta 2-J delta 1 and V gamma 9-J gamma 1.2 rearrangements of peripheral T-lymphocytes showed high frequencies of leucine and valine at a fixed position in the V delta 2-J delta 1 junctional regions. This phenomenon remained unnoticed in the numerous published junctional regions for over several years. Because comparable preferential motifs might also occur in junctional regions of other T-cell populations in health and disease, more precise analysis of junctional region diversity is needed. For this reason we describe general guidelines for identification of the various elements in TcR junctional regions: D-gene-derived nucleotides (in case of TcR-beta and TcR-delta genes), P-region nucleotides, N-region nucleotides, and deletion of nucleotides by trimming of the rearranged gene segments. In addition, we summarized the known genomic germline sequences of rearranging TcR gene segments, which are necessary for proper application of the general guidelines. Subsequent analysis of the majority of published TcR junctional regions, allowed us to determine the composition and average insertion and deletion of nucleotides in genomic junctional regions. Because the protein junctional region instead of the genomic junctional region determines the actual specificity of TcR chains, the amino acid composition of the protein junctional regions of different types of TcR gene rearrangements was determined. This revealed some unexpected characteristics, such as the virtual absence of cysteine in all functional TcR junctional regions and increased or decreased frequencies of particular amino acid residues in specific TcR junctional regions. Application of the guidelines in combination with the summarized TcR germline sequences may contribute to uniformity in the analysis of junctional regions and may lead to important information concerning TcR specificity.