The phosphorylated acidic glycoprotein osteopontin is present in the extracellular matrix of atherosclerotic plaques and the wall of injured but not normal arteries. To determine if osteopontin could serve as a substrate for platelet adhesion, we measured the adherence of resting and agonist-stimulated human platelets to immobilized recombinant human osteopontin. Agonist-stimulated but not resting platelets bound to osteopontin by a process that was mediated primarily by alphavbeta3. alphavbeta3-mediated adherence occurred at physiologic concentrations of calcium and was inhibited by an alphavbeta3-selective cyclic peptide. Assays using phorbol myristate acetate-stimulated transfected B lymphocytes expressing both alphavbeta3 and alphaIIbbeta3 confirmed that activated alphavbeta3 not activated alphaIIbbeta3 was responsible for the cellular adherence we measured. These studies indicate that alphavbeta3 can reside on the cell surface in an inactive state and can be converted to a ligand binding conformation by cellular agonists. Moreover, they suggest that platelet adherence to osteopontin mediated by activated alphavbeta3 could play a role in anchoring platelets to disrupted atherosclerotic plaques and the walls of injured arteries. By inhibiting alphavbeta3 function, it may be possible to inhibit platelet-mediated vascular occlusion with a minimal effect on primary hemostasis.