Recently, we reported that vimentin-type intermediate filaments, in addition to microfilaments, associate with alphavbeta3 integrin-positive focal contacts in endothelial cells. To gain insight into intermediate filament-focal contact interaction, we induced expression of yellow fluorescent protein (YFP)-integrin beta3 and cyan fluorescent protein (CFP)-vimentin protein in endothelial cells. At least 50% of the YFP-beta3 integrin-labeled focal contacts associated with CFP-labeled vimentin intermediate filaments in live cells. Moreover, focal contacts and intermediate filaments moved in concert in the plane of the membrane and assembling focal contacts were sites of vimentin filament assembly. When endothelial cells were subjected to flow, large focal contacts assembled and associated with thick vimentin bundles. These large focal contacts showed minimal dynamic activity. Cells in which vimentin expression had been inhibited by RNA interference assembled smaller than normal focal contacts. More dramatically, such cells showed decreased adhesion to the substratum. These data provide evidence that the vimentin cytoskeleton regulates focal contact size and helps stabilize cell-matrix adhesions in endothelial cells.