Integrins play a key role in regulating endothelial cell survival, migration and differentiated function during angiogenic blood-vessel remodeling. Integrin-linked kinase (ILK) is a multidomain protein that interacts with the cytoplasmic tail of integrin beta subunits and is thought to participate in integrin-mediated signal transduction. We report here that attenuation of ILK expression in cultured bovine aortic endothelial cells by RNA interference had marked effects on surface distribution of alpha5beta1 integrin and the organization of cell-matrix adhesions characterized by the disappearance of fibrillar (3D-like) adhesions that are rich in alpha5beta1 and paxillin, and associated fibrillar fibronectin matrix. This defect was not caused by a decrease in fibronectin mRNA levels or by intracellular retention of the protein. Adhesion to surface-adsorbed matrix proteins based on beta1 and beta3 integrin was enhanced following ILK depletion, whereas cell spreading, migration and multilayer alignment into capillary-like structures on Matrigel were impaired. We conclude that ILK is an important regulator of the endothelial phenotype and vascular network formation by directing the assembly and/or maturation of alpha5beta1-competent matrix-forming adhesions.