The divalent cation Mn²⁺ and the reducing agent dithiothreitol directly shift integrins from their inactive to their active states. We used transmission electron microscopy and laser tweezers-based force spectroscopy to determine whether structural rearrangements induced by these agents in the integrin αIIbβ3 correlate with its ability to bind fibrinogen. Mn²⁺ increased the probability of specific fibrinogen-αIIbβ3 interactions nearly 20-fold in platelets, and both Mn²⁺ and dithiothreitol increased the probability more than 2-fold using purified proteins. Of 3 αIIbβ3 conformations, closed with stalks touching, open with stalks separated, and globular without visible stalks, Mn²⁺ and dithiothreitol induced a significant increase in the proportion of open structures, as well as structural changes in the αIIbβ3 headpiece. Mn²⁺ also increased the number of complexes between fibrinogen and purified αIIbβ3 molecules, all of which were in the open conformation. Finally, Mn²⁺ induced the formation of αIIbβ3 clusters that resulted from interactions exclusively involving the distal ends of the stalks. These results indicate that there is a direct correlation between αIIbβ3 activation and the overall conformation of the molecule. Further, they are consistent with the presence of a linked equilibrium between single inactive and single active αIIbβ3 molecules and active αIIbβ3 clusters. (Blood. 2004;104:3979-3985)