T-cell receptor (TCR) alpha-chain (TCR alpha) and beta-chain (TCR beta) genes are well characterized in mammals, while only TCR beta genes have been identified in other vertebrates. To identify avian TCR alpha genes, we used monoclonal anti-CD3 antibodies to isolate chicken TCR alpha for peptide sequence analysis. Degenerate oligonucleotide probes were then used to isolate a candidate TCR alpha cDNA clone that hybridized with a 1.7-kb mRNA species present only in alpha beta T cells and in tissues populated by these cells. Southern blot analysis revealed gene rearrangement in thymocytes and alpha beta T-cell lines. The TCR alpha cDNA candidate encoded an open reading frame of 275 amino acids, the predicted variable (V)-, joining (J)-, and constant (C)-region amino acid sequences of which shared approximately 40%, 60%, and 25% homology with corresponding mammalian sequences. A single C alpha gene and approximately 25 V alpha genes were identified by using region-specific probes. The V alpha cDNA probe isolated from a V beta 1+ cell line reacted with transcripts from one of five V beta 2+ cell lines, suggesting shared use of V alpha genes by V beta 1+ and V beta 2+ T cells and the existence of other V alpha gene families. A genomic V alpha sequence was flanked by classical recombination signal sequences but, unlike previously defined V genes, the leader and V alpha region were encoded by a single exon. The data indicate evolutionary conservation of the basic TCR alpha gene structure in birds and mammals.