We have investigated receptor function and epitope expression of recombinant αIIbβ3mutated at Cys177or Cys273in the I-like domain as well as Cys598, located in the fourth repeat of the membrane-proximal cysteine-rich region and mutated in a Glanzmann's thrombasthenia type II patient. The β3mutants β3C177A, β3C273A, and β3C598Y exhibited a decreased electrophoretic mobility in SDS-polyacrylamide gel electrophoresis under nonreducing conditions, confirming the disruption of the respective disulfide loops. Despite reduced surface expression, the αIIbβ3C177A, αIIbβ3C273A, and αIIbβ3C598Y receptors mediated cell adhesion to immobilized fibrinogen and translocated into focal adhesion plaques. The β3C598Y mutation, but not the β3C177A or β3C273A mutations, induced spontaneous binding of the ligand mimetic monoclonal antibody PAC-1, while the β3C177A and β3C273A mutants exhibited reduced complex stability in the absence of Ca2+. Epitope mapping of function-blocking monoclonal antibodies (mAbs) allowed the identification of two distinct subgroups; mAbs A2A9, pl2–46, 10E5, and P256 did not interact with αIIbβ3C273A and bound only weakly to αIIbβ3C177A, while mAbs AP2, LM609 and 7E3 bound normally to mutant αIIbβ3C273A, but interacted only weakly with mutant αIIbβ3C177A. Furthermore, a cryptic epitope recognized by mAb 4D10G3 and not exposed on wild type αIIbβ3became accessible only on mutant αIIbβ3C177A and was mapped to the 60-kDa chymotrypsin fragment of β3. Finally, the ligand-induced binding site (LIBS) epitopes AP5, D3, LIBS1, and LIBS2 were spontaneously expressed on all three mutants independent of RGDS or dithiothreitol treatment. Our results provide evidence that disruption of a single cysteine disulfide bond in the cysteine-rich repeat domain, but not in the I-like domain, activates integrin αIIbβ3. In contrast, disruption of each of the disulfide bonds in the two long insertions of the I-like domain predicted to be in close contact with the α subunit β-propeller domain affect the stability of the αIIbβ3heterodimer and inhibit complex-specific mAb binding without affecting the RGD binding capacity of the metal ion-dependent adhesion site-like domain.