Coadsorption of high-affinity avidin with lower affinity cell adhesion protein fibronectin has been shown to significantly augment short-term (1 h) adhesion and spreading of endothelial cells; however, the longer term persistence of avidin binding and its effect on endothelial cell adhesion have not been addressed. In this study, the presence of avidin-biotin bonds 24 h after cell adhesion to the dual ligand surfaces was verified by laser confocal microscopy of a fluorescent avidin analog, streptavidin. Total internal reflection microscopy showed that the focal contact area, focal contact density, and cell spreading all increased significantly at 24 h compared to fibronectin-treated control surfaces. Focal contact area was identical when measured with cells that were labeled with either the fluorescent streptavidin or a carbocyanine dye incorporated in the cell membrane. Confocal images of stress fibers formed in cells adherent to dual ligand surfaces after 24 h were thicker and more numerous compared to cells adherent to fibronectin controls. The results indicate that 24 h after initial attachment avidin-biotin is localized to focal contacts on the basal surface and affects cell spreading, actin filament organization, and focal contact density.