Many integrin adhesion receptors bind ligands containing the Arg-Gly-Asp (RGD) peptide motif. Most integrins exhibit considerable specificity for particular ligands and can distinguish among the many conformations of RGD. In this study we identify the domain of the integrin  subunit involved in determining ligand binding specificity. Chimeras of 3 and 5, the most homologous integrin  subunits, were expressed with ␣v on the surface of human 293 cells. The ligand binding phenotype of each chimera was assessed using the ligands Fab-9 and fibrinogen, both of which have a binding preference for ␣v3. The results of the study show that when exons C and D of the 3 subunit (residues 95-233) are substituted into 5, the chimera gained the ability to bind Fab-9 with an affinity close to that of wild-type ␣v3. This chimera was able to mediate cell adhesion to fibrinogen. Furthermore, the swap of only a 39-residue segment of this larger domain, 3 residues 164 -202, into the backbone of 5 enabled the chimeric integrin to bind soluble Fab-9. This small domain is highly divergent among the integrin  subunits, suggesting that it may play a role in determining ligand selection by all integrins.Integrins are transmembrane ␣ heterodimers that are responsible for most of the physical contacts between cells and the extracellular matrix. Integrins are involved in a number of tissue remodeling events including embryogenesis, angiogenesis, wound repair, and bone resorption (1-5). Integrins are also causally linked to pathological conditions such as tumor progression, thrombosis, inflammation, and osteoporosis (6 -8).The integrin protein family contains 13 ␣ and 9  subunits. These subunits can cross-pair to form heterodimers with different expression patterns and distinct ligand binding profiles (9, 10). Each integrin binds to only a limited series of ligands, ensuring that cell adhesion and migration are precisely regulated. Such precision is key to tissue remodeling.Based on their ligand recognition specificity, integrins can be divided into two broad sub-families. About one-half of the integrins bind to ligands that lack any consensus binding motif.The remaining integrins bind to the RGD 1 consensus sequence. Importantly though, even the integrins that bind to RGD display a great deal of specificity for matrix ligands (11,12). Some of this specificity is determined by contacts that are ancillary to the RGD sequence (13,14), but much of the specificity is determined by the shape of the RGD motif and the residues in its immediate vicinity. Thus, to understand the structural basis of matrix selection, it is important to understand how integrins distinguish the many conformations of RGD.The goal of the present study was to identify domain(s) within the integrin  subunit that confer ligand binding specificity. As a model system, we compared the ligand binding domains of the two most homologous integrins, ␣v3 and ␣v5. The 3 and 5 subunits have 56% identity at the amino acid level (15, 16). Although both ␣v3 and ␣v5 recogn...