Eight integrin ?-? heterodimers recognize ligands with an Arg-Gly-Asp (RGD) motif. However, the structural mechanism by which integrins differentiate among extracellular proteins with RGD motifs is not understood. Here, crystal structures, mutations and peptide-affinity measurements show that ?V?6 binds with high affinity to a RGDLXXL/I motif within the prodomains of TGF-?1 and TGF-?3. The LXXL/I motif forms an amphipathic ?-helix that binds in a hydrophobic pocket in the ?6 subunit. Elucidation of the basis for ligand binding specificity by the integrin ? subunit reveals contributions by three different ?I-domain loops, which we designate specificity-determining loops (SDLs) 1, 2 and 3. Variation in a pair of single key residues in SDL1 and SDL3 correlates with the variation of the entire ? subunit in integrin evolution, thus suggesting a paradigmatic role in overall ?-subunit function.