The integrin α9β1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin α4β1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether α9β1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that α9β1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-α. Furthermore, we show that α9β1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, α9β1 and α4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for α9β1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.