A mAb recognizing a 40- to 44-kDa monomeric molecule on the surface of chicken T cells was used to screen a cDNA expression library made from Con A-stimulated chicken spleen cells. The sequence of the cDNA obtained encoded a molecule having 50% amino acid sequence identity with mammalian CD28, but the cysteine residue involved in the inter-chain bridge of the mammalian CD28 homodimer was not conserved in the chicken sequence. The molecule produced in transfected COS-7 cells was also recognized by another mAb that had previously been thought to recognize an avian homologue of CD2. The sequence data establish that this molecule is a homologue of mammalian CD28 in the strict evolutionary sense.