Horse (Equus caballus) T-cell receptor alpha (TCRA), gamma (TCRG), and delta (TCRD) chain genes were isolated from a cDNA library and characterized. Five unique TCRAV families, including four full-length sequences, five distinct TCRAJ genes, and a single TCRAC gene were identified. TCRAV genes had closest homology with human sequences and least similarity to rat genes. Among eight horse TCRG genes, two distinct constant region genes with considerable variation in the connecting region were identified, but no variable or joining genes were present. Southern blot hybridization confirmed the presence of at least two TCRGC genes and indicated that the vast majority of horse alpha beta T cells rearrange either one or both TCRG alleles. Analysis of horse TCRD genes revealed the presence of eight unique TCRDV genes representing seven families, each having closest nucleotide homology with sheep sequences. Six unique TCRDJ genes were isolated; however, four of these sequences differed by only one base pair and thus likely represented alleles of a single gene. One horse TCRDC gene was present among fifteen clones analyzed and, based on Southern blot hybridizations, was deleted in polyclonal alpha beta T-cell populations, indicating that the TCRD locus is probably located within the TCRA locus as in other species. Polymerase chain reaction using horse-specific primers for the detection of TCRAC and TCRDC gene expression indicated that gamma delta T cells are located at numerous sites throughout the body, and with the exception of bone marrow where only TCRAC transcripts were detected, are closely associated with alpha beta T cells. This finding indicates that these two T-cell populations may be functionally interactive.