During acute inflammatory processes, β₂ and β₁ integrins sequentially mediate leukocyte recruitment into extravascular tissues. We studied the influence of VLA-4 (very late antigen-4) (₄β₁) engagement on β₂ integrin activation-dependent cell-to-cell adhesion. Ligation of VLA-4 by the soluble chimera fusion product vascular cell adhesion molecule-1 (VCAM-1)–Fc or by 2 anti-CD29 (β₁ chain) monoclonal antibodies (mAb) rapidly induced adhesion of myelomonocytic cells (HL60, U937) to human umbilical vein endothelial cells (HUVECs). Cell adhesion was mediated via β₂ integrin (LFA-1 and Mac-1) activation: induced adhesion to HUVECs was inhibited by blocking mAbs anti-CD18 (70%-90%), anti-CD11a (50%-60%), or anti-CD11b (60%-70%). Adhesion to immobilized ligands of β₂ integrins (intercellular adhesion molecule-1 [ICAM-1], fibrinogen, keyhole limpet hemocyanin) as well as to ICAM-1–transfected Chinese hamster ovary cells, but not to ligands of β₁ integrins (VCAM-1, fibronectin, laminin, and collagen), was augmented. VCAM-1–Fc binding provoked the expression of the activation-dependent epitope CBRM1/5 of Mac-1 on leukocytes. Clustering of VLA-4 through dimeric VCAM-1–Fc was required for β₂ integrin activation and induction of cell adhesion, whereas monovalent VCAM-1 or Fab fragments of anti-β₁ integrin mAb were ineffective. Activation of β₂ integrins by ₄β₁ integrin ligation (VCAM-1–Fc or anti-β₁ mAb) required the presence of urokinase receptor (uPAR) on leukocytic cells, because the removal of uPAR from the cell surface by phosphatidylinositol-specific phospholipase C reduced cell adhesion to less than 40%. Adhesion was reconstituted when soluble recombinant uPAR was allowed to reassociate with the cells. Finally, VLA-4 engagement by VCAM-1–Fc or anti-β₁ integrin mAb induced uPAR-dependent adhesion to immobilized vitronectin as well. These results elucidate a novel activation pathway of β₂ integrin–dependent cell-to-cell adhesion that requires ₄β₁ integrin ligation for initiation and uPAR as activation transducer.