The α2β1 integrin serves as a cell surface collagen or collagen/laminin receptor. Binding of the integrin to its ligands is largely mediated by the α2 subunit I domain and requires the presence of divalent cations. Terbium ion (Tb3+), a fluorescent trivalent cation that often binds divalent cation-binding sites on proteins, supported binding of the I domain to collagen with half-maximal binding occurring at 5.2 ± 1.7 μM Tb3+. By fluorescence resonance energy transfer spectroscopy, Tb3+ showed specific and saturable binding to the recombinant I domain with a K(d) of 27 ± 4 μM. Although both Mg2+ and Mn2+ were capable of quenching Tb3+ fluorescence, Mn2+ was much more effective than M2+. The α2β1 integrin also binds the pro-α1(I) collagen carboxyl-terminal propeptide in a Mg2+-dependent manner via the I domain. Recombinant propeptide was used to examine the effect of ligand on the Tb3+ binding properties of the α2 integrin I domain. As propeptide bound to the I domain, Tb3+ fluorescence progressively diminished suggesting that as ligand binds to the I domain, either Tb3+ is displaced or its fluorescence is quenched. Consistent with the former possibility, little dissociation of collagen-bound I domain occurred upon the addition of EDTA and subsequent incubation. These data support a model in which (1) the divalent cation is required for initial ligand-binding activity of the I domain and (2) ligand binding results in subsequent metal ion displacement to generate a metal-free I domain-ligand complex.