Leukocyte recruitment plays a major role in the immune response to infectious pathogens, as well as during inflammatory and autoimmune disorders. The process of leukocyte extravasation from the blood requires a complex cascade of adhesive events between the leukocytes and the endothelium, including initial leukocyte rolling, adhesion, and finally transendothelial migration. Current research in this area aims to identify the key leukocyte subsets that initiate a given disease and to identify the trafficking molecule(s) that will most specifically inhibit those cells. Herein we demonstrate that tumor necrosis factor (TNF)alpha activates the integrin alpha(5)beta(1) without altering total expression levels of beta(1) integrin on human umbilical vein endothelial cells. Moreover, our studies suggest that TNFalpha-induced beta(1) activation is dependent on sphingosine kinase-1, but independent of the sphingosine-1-phosphate family of G protein-coupled receptors. We also show, using a parallel plate flow chamber assay, that neutrophil adhesion to TNFalpha-activated endothelium can be attenuated by blocking alpha(5)beta(1) or its ligand angiopoietin-2. These observations add new complexities that broaden the accepted concept of cellular trafficking with neutrophil adhesion to TNFalpha activated endothelial cells being sphingosine kinase-1, alpha(5)beta(1), and angiopoietin-2 dependent. Moreover, this work supports the notion that sphingosine kinase-1 may be the single target required for an effective broad spectrum approach to combat inflammation and immune disorders.