We have isolated and identified all four TCR alpha, beta, gamma, and delta cDNAs and genomic clones from a Japanese flounder leukocyte cDNA library and bacterial artificial chromosomal genomic library. Numerous TCR transcripts were sequenced to examine the variability against antigenic peptide, and were shown hypervariability on their complementarity-determining region 3 (CDR3) loops. Among CDR3s, CDR3 delta showed a long and broad length distribution, indicating greater similarity to that of Ig. From cDNA sequences and genomic gene analysis of each chain, we found that flounder TCR beta, gamma, and delta have two different C gene segments, while the TCR alpha C region exists as a single segment. The flounder C gammas and C deltas showed different lengths in the connecting peptide (CP) region between the different types of polypeptides. The C delta 1 gene consists of two exons, one that encodes an extracellular Ig-like domain (exon 1) and the other that encodes either a very short or possibly a lacking CP region, a transmembrane region, and a cytoplasmic tail (exon 2); these are located within TCR alpha gene locus. Southern blot analysis, using the bacterial artificial chromosomal genomic DNA clones, revealed that the C delta 2 gene segment, which has a long CP region and different genomic organization to the C delta 1 gene, exists on same gene locus as the TCR gamma-chain. This suggests that the flounder possesses very unique genomic DNA organization and gene loci for TCR, C alpha/C delta 1, and C gamma/C delta 2.