Polymorphonuclear leukocytes (PMN) respond to tumor necrosis factor (TNF) with a respiratory burst (RB) only after adherence to surfaces coated with extracellular matrix proteins such as fibronectin and fibrinogen (permissive substrates) but not with others such as laminin or collagen (nonpermissive substrates). As PMN adherence to both types of surfaces is dependent on β₂ integrins, we investigated the molecular basis of the different metabolic response to TNF. In particular, we evaluated the relative role of each β₂ integrin (α_Lβ₂, α_Mβ₂, and α_Xβ₂) in adherence and O₂⁻ production of PMN residing on fibronectin- and laminin-coated surfaces, which were considered as models of permissive and nonpermissive surfaces, respectively. By using α chain-specific monoclonal antibodies (mAb), we show that α_Mβ₂ and α_Xβ₂ mediate adherence to fibronectin and laminin; α_Lβ₂ is not involved in adherence to laminin and has only a minimal contribution in adherence to fibronectin. Furthermore, production of O₂⁻ in response to TNF was induced by immobilized anti-α_Lβ₂ but not anti-α_Mβ₂ or anti-α_Xβ₂ mAb. A strong correlation was also found between expression of α_Lβ₂ and TNF-induced RB on fibronectin. Lastly, PMN responded to TNF on laminin with a RB after the inclusion of α_L-specific mAb in the laminin coat. Thus, we conclude that TNF-induced RB by PMN residing on fibronectin is mediated by α_Lβ₂ and that α_Mβ₂ and α_Xβ₂ are likely to play an ancillary role to the signaling activity of α_Lβ₂ by facilitating its recruitment to sites of adherence. The nonpermissiveness of laminin appears to be a consequence of its inability to act as a ligand for α_Lβ₂.