In this study, the effects of the streptavidin(SA)-biotin ligands on the amount of endothelium-derived nitric oxide synthase (eNOS) were quantified. The higher-affinity SA-biotin ligands were previously introduced to help promote the initial endothelial cell attachment to fibronectin coated surfaces. To validate their applications, SA-biotin ligands must be demonstrated to have minimal adverse effects on the eNOS activity, a process that is crucial to the vasotone regulation. Results in this study suggest that not only did the SA-biotin ligands have no adverse effects on the eNOS activity, they significantly promoted it. We speculate that this experimental observation is likely due to the enhancement of the different cell adhesion parameters, including the cellular spreading, cell adhesion strength, as well as the formation of focal contacts. Because of the improvement of these parameters, the cells were able to attach onto the surfaces more rapidly and firmly, thereby enabling the formation of a stable and rapidly crosslinked cell cytoskeletal network. The development and formation of such stable cytoskeleton is crucial for the shear stress signals to transmit through the cell and to eventually activate the different intracellular events that are required to promote the eNOS activity.