Recent work has shown that osteopontin expression is upregulated at sites of cardiovascular injury. It has been hypothesized that osteopontin provides an adhesive matrix for endothelial and smooth muscle cells during remodeling of the vascular wall following injury. Osteopontin has also been found to be synthesized by monocytes and macrophages within injury sites. Here, we present data showing that osteopontin can promote leukocyte adhesion through the α₄β₁ integrin. In the presence of physiologic concentrations of Mg²⁺ and Ca²⁺, osteopontin purified from bovine milk promoted cell-substrate adhesion of HL-60 and Ramos cells, two model leukocyte cell lines. As with other adhesive ligands, adhesion to osteopontin required leukocyte activation. Under these conditions, no adhesion to control substrates such as bovine serum albumin was observed. Leukocyte adhesion was inhibited by anti-integrin antibodies directed at either the α₄ or β₁ integrin subunits but not by control antibodies directed to other integrins. Further adhesion experiments revealed that leukocyte binding to osteopontin was completely inhibited by an α₄β₁-binding peptide containing the leucine-aspartate-valine (LDV) sequence, while a control, non-binding peptide containing leucine-glutamate-valine (LEV) had minimal effects. Affinity chromatography using either surface labeled HL-60 or Ramos cell extracts revealed that the α₄β₁ integrin specifically bound to osteopontin. Immunoprecipitation of eluted fractions from these columns positively identified the α₄β₁ integrin. In order to localize potential α₄β₁-binding sites within osteopontin, the protein was proteolytically cleaved with thrombin. A 30 kDa N-terminal osteopontin fragment purified using fast protein liquid chromatography promoted α₄β₁ dependent leukocyte adhesion in a manner similar to that of the intact protein. These data collectively demonstrate that the α₄β₁ integrin is a new adhesion receptor for osteopontin and that an α₄β₁ binding site exists in the NH₂-terminal thrombin fragment of osteopontin.