Here we use a model RGD-containing ligand to study how Ca2+ and Mg2+ regulate ligand binding to beta3-integrins. Fab-9, an antibody that contains an optimized RGD loop in its antigen binding site (Barbas, C. F., Languino, L., and Smith, J. W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10003-10007), was used as the model ligand. Across a physiologic range of Mg2+, Fab-9 bound to both alphavbeta3 and alphaIIbbeta3 with a monophasic binding isotherm. Across the same range of Ca2+, the binding of Fab-9 to the beta3-integrins was biphasic. Low concentrations of Ca2+ (microM) promoted the binding of Fab-9. Higher concentrations of Ca2+ (m) blocked Fab-9 binding. These data suggest that Ca2+ binds to two distinct classes of sites on the beta3-integrins, with the low affinity Ca2+ binding site(s) being an inhibitory site. We designate this inhibitory site(s) as the I site. Further biochemical characterization showed that the I site has the following characteristics: 1) it is specific for Ca2+; 2) it is allosteric to the ligand binding site; 3) its occupation increases the dissociation rate between integrin and RGD ligand; and 4) occupation of the I site can induce cellular deadhesion.