Adhesion is known to prime neutrophils for physiological activation in response to cytokines and other stimuli. We have employed the technique of receptor cross-linking to study the potential role of CD18, the common β-subunit of the β₂-integrin family of adhesion molecules, in the regulation of the respiratory burst, as measured by luminol-enhanced chemiluminescence and iodination, in human neutrophils. CD18 cross-linking primed neutrophils to activate the respiratory burst after stimulation with tumor necrosis factor α (TNF-α) (100 units/mL), formyl-methionyl-leucyl-phenylalanine (fMLP) (1 μM), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (1 μg/mL), but not granulocyte colony-stimulating factor (G-CSF) (1 μg/mL), interferon-γ (IFN-γ) (100 U/mL), or phorbol myristate acetate (100 nM). The maximal rate of chemiluminescence induced by fMLP, TNF-α, and GM-CSF was enhanced 8-, 6-, and 1.5-fold, respectively, following CD18 cross-linking. Priming of the respiratory burst by direct engagement of CD18 was confirmed in neutrophil-mediated iodination experiments, where iodination induced by TNF-α, fMLP, and GM-CSF was increased 15-, 20-, and 7-fold, respectively, by CD18 cross-linking. Immunoblot experiments demonstrated that TNF-α-induced tyrosine phosphorylation was both accelerated and more intense in neutrophils after cross-linking of CD18. Major tyrosine phosphoprotein products include proteins with approximate molecular masses of 40, 70, and 110 kDa. Genistein (50μM), a selective tyrosine kinase inhibitor, reduced the TNF-α-stimulated respiratory burst by >80% whether or not CD18 was cross-linked. These results affirm the importance of CD18 in adhesion-dependent priming of neutrophil functions and demonstrate that CD18 engagement per se is sufficient to prime neutrophils for cytokine-induced signal transduction mediated by tyrosine phosphorylation.