Compared with human platelets, rodent platelets are less responsive to peptides and peptidomimetics containing an arginine-glycine-aspartic acid (RGD) motif. Using chimeric human-rat alphaIIbbeta3 molecules, we found that this difference in Arg-Gly-Asp-Ser (RGDS) sensitivity was the result of amino acid substitutions at residues 157, 159, and 162 in the W3:4-1 loop and an Asp-His replacement at residue 232 in the W4:4-1 loop of the alphaIIb beta propeller. Introducing the entire rat W3:4-1 and W4:4-1 loops into human alphaIIbbeta3 also decreased the inhibitory effect of the disintegrins, echistatin and eristostatin, and the alphaIIbbeta3 antagonists, tirofiban and eptifibatide, on fibrinogen binding, whereas the specific point mutations did not. This suggests that RGDS interacts with alphaIIb in a different manner than with these small molecules. None of these species-based substitutions affected the ability of alphaIIbbeta3 to interact with RGD-containing macromolecules. Thus, human von Willebrand factor contains an RGD motif and binds equally well to adenosine diphosphate-stimulated human and rodent platelets, implying that other motifs are responsible for maintaining ligand binding affinity. Many venoms contain RGD-based toxins. Our data suggest that these species amino acids differences in the alphaIIb beta-propeller represent an evolutionary response by rodents to maintain hemostasis while concurrently protecting against RGD-containing toxins.