Integrins alpha IIb beta 3 and alpha V beta 3 mediate numerous cell-matrix and cell-cell contacts. Both integrins contain multiple divalent cation-binding motifs that regulate ligand binding. Here, we elucidate a major difference in the regulation of alpha IIb beta 3 and alpha V beta 3 by divalent ions. Fibrinogen binding to alpha IIb beta 3 in Ca(2+)-containing buffer is rapid, with an apparent association rate constant (k1app) of 8.2 x 10(5) M-1 s-1, but Ca2+ does not support association between fibrinogen and alpha V beta 3. Interestingly, Mn2+ supports fibrinogen binding to both integrins, albeit with a relatively slow association rate (k1app = 10(4) M-1 s-1). This influence of divalent ions on ligand association rates accounts for the opposite divalent ion requirements for platelet aggregation and tumor cell adhesion to fibrinogen. Furthermore, the regulation of fibrinogen binding to alpha V beta 3 is complex when both Ca2+ and Mn2+ are present. Physiological concentrations of Ca2+ completely ablated adhesion. Kinetic analysis demonstrated that Ca2+ is a mixed-type inhibitor of Mn(2+)-supported fibrinogen binding to alpha V beta 3. Consequently, the data presented here suggest a mechanism in which two separate cation-binding sites regulate ligand binding to beta 3-integrins.