The main interface of the 2 subunits of platelet integrin αIIbβ3 comprises the β-propeller domain of αIIb and the βA domain of β3. In the center of the β-propeller, several aromatic residues interact by cation-π and hydrophobic bonds with Arg261 of βA. In this study, we substituted αIIb-Trp110 or β3-Arg261 by residues abundant in other α or β subunits at corresponding locations and expressed them in baby hamster kidney cells along with normal β3or αIIb, respectively. These mutant cells displayed normal surface expression and fibrinogen binding but grossly impaired outside-in signaling-related functions: adhesion to immobilized fibrinogen, cell spreading, focal adhesion kinase phosphorylation, clot retraction, and reduced αIIbβ3 stability in EDTA (ethylenediaminetetraacetic acid). Expression of mutants with substitutions of Arg261 in β3 by alanine or lysine with normal αv yielded normal surface expression of αvβ3 and soluble fibrinogen binding as well as normal outside-in signaling-related functions, contrasting findings for αIIbβ3. Structural analysis of αIIbβ3 and αvβ3 revealed that αvβ3 has several strong interactions between αv and β3 subunits that are missing in αIIbβ3. Together, these findings indicate that the interaction between Trp110 of αIIb and Arg261 of β3 is critical for αIIbβ3 integrity and outside-in signaling-related functions.