We have cloned the first CD8α gene from an ectothermic source using a degenerate primer for Ig superfamily V domains. Similar to homologues in higher vertebrates, the rainbow trout CD8α gene encodes a 204-aa mature protein composed of two extracellular domains including an Ig superfamily V domain and hinge region. Differing from mammalian CD8α V domains, lower vertebrate (trout and chicken) sequences do not contain the extra cysteine residue (C strand) involved in the abnormal intrachain disulfide bridging within the CD8α V domain of mice and rats. The trout membrane proximal hinge region contains the two essential cysteine residues involved in CD8 dimerization (αα or αβ) and threonine, serine, and proline residues which may be involved in multiple O-linked glycosylation events. Although the transmembrane region is well conserved in all CD8α sequences analyzed to date, the putative trout cytoplasmic region differs and, in fact, lacks the consensus p56 lck motif common to other CD8α sequences. We then determined that the trout CD8α genomic structure is similar to that of humans (six exons) but differs from that of mice (five exons). Additionally, Northern blotting and RT-PCR demonstrate that trout CD8α is expressed at high levels within the thymus and at weaker levels in the spleen, kidney, intestine, and peripheral blood leukocytes. Finally, we show that trout CD8α can be expressed on the surface of cells via transfection. Together, our results demonstrate that the basic structure and expression of CD8α has been maintained for more than 400 million years of evolution.