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To further identify amino acid domains involved in the ligand binding specificity of αIIbβ3, chimeras of the conserved calcium binding domains of αIIband the α subunit of the fibronectin receptor α5β1were constructed. Chimeras that replaced all four calcium binding domains, replaced all but the second calcium binding domain of αIIbwith those of α5, or deleted all four calcium binding domains were synthesized but not expressed on the cell surface. Additional chimeras exchanged subsets or all of the variant amino acids in the second calcium binding domain, a region implicated in ligand binding. Cell surface expression of each second calcium binding domain mutant complexed with β3was observed. Each second calcium binding domain mutant was able to 1) bind to immobilized fibrinogen, 2) form fibrinogen-dependent aggregates after treatment with dithiothreitol, and 3) bind the activation-dependent antibody PAC1 after LIBS 6 treatment. Soluble fibrinogen binding studies suggested that there were only small changes in either the KdorBmaxof any mutant. We conclude that chimeras of αIIbcontaining the second calcium binding domain sequences of α5are capable of complexing with β3, that the complexes are expressed on the cell surface, and that mutant complexes are capable of binding both immobilized and soluble fibrinogen, suggesting that the second calcium binding domain does not determine ligand binding specificity.