An unbiased genetic approach was used to identify a specific amino acid residue in the _IIb subunit important for the ligand binding function of the integrin _IIbβ. Chemically mutagenized cells were selected by flow cytometry based on their inability to bind the ligand mimetic antibody PAC1 and a cell line containing a single amino acid substitution in _IIb at position 224 (D→V) was identified. Although well expressed on the surface of transfected cells, _IIbD224Vβ₃ as well as _IIbD224Aβ₃ did not bind _IIbβ₃-specific ligands or a RGD peptide, a ligand shared in common with _vβ₃. Insertion of exon 5 of _IIb, residues G193-W235, into the backbone of the _v subunit did not enable the chimeric receptor to bind _IIbβ₃-specific ligands. However, the chimeric receptor was still capable of binding to a RGD affinity matrix. _IIbD224 is not well conserved among other integrin  subunits and is located in a region of significant variability. In addition, amino acid D224 lies within a predicted loop of the recently proposed β-propeller model for integrin  subunits and is adjacent to a loop containing amino acid residues previously implicated in receptor function. These data support a role for this region in ligand binding function of the _IIbβ₃ receptor.