The binding of orthosteric ligands to integrins requires the presence of divalent metal cations bound to metal ion-binding sites located in the I domains of the integrin α and β subunits. In this study the influence of the type and concentration of divalent metal cation present was investigated on a single arginyl-glycinyl-aspartic acid (RGD) ligand across the αv integrin sub-family and single αv integrin (αvβ6) with different ligands. These relationships were determined using radioligand binding studies completed with [³H] ligands and purified αv integrin protein preparations. The binding of [³H]compound 1 to the RGD site on individual αv integrins demonstrated a unique profile in relation to the type and concentration of divalent metal cation present. The use of physiological concentrations of Mg²⁺ and Ca²⁺ in simulated lung fluid altered the αv integrin selectivity profile of [³H]compound 1 in terms of affinity and the level of receptor occupancy. In addition, different RGD ligands for the αvβ6 integrin behaved differently under the same divalent metal cation conditions. In conclusion, this study demonstrates the need to determine the individual relationship between RGD ligands and the integrins they may engage in vivo, especially when determining selectivity profiles for potential RGD-mimetic small molecule therapeutics, with organ and disease state also considered.