Osteopontin is an RGD-containing glycoprotein that mediates adhesion and migration of a variety of different cell types. We recently showed that a recombinant osteopontin fragment that was expected to be formed following thrombin cleavage was not only biologically active, but also could support α₉β₁-mediated adhesion, an activity not found in the full-length molecule. In this study we defined binding sites on the N-terminal osteopontin fragment important for α₉β₁-mediated cell interactions. In addition to adhesion, we showed that α₉β₁could mediate cell migration, a function not previously identified for this integrin. Using site-directed mutagenesis, we made specific mutations in the RGD region of the N-terminal osteopontin fragment. Mutation of RGD to RGE resulted in a 50% decrease in cell adhesion. The residual binding to the RGE mutant could be blocked by α₉and β₁antibodies. Adhesion to the RGE mutant was due to residual recognition of the RGE site by α₉β₁since mutants containing more drastic mutations in the RGD domain achieved by mutating RGD to RAA or by eliminating the RGD completely failed to support cell adhesion and migration. In contrast, α₉β₁-mediated adhesion to tenascin was RGD independent. These data demonstrate that α₉β₁is one of the few integrin receptors that can interact with two distinct RGD-containing ligands through different adhesive domains.