Alpha-actinins are critical components of the actin cytoskeleton. Here we show that -actinins serve another important biological function by binding to and competitively inhibiting calcium-dependent activation of endothelial NOS (eNOS). -actinin-2 was found to associate with eNOS in a yeast two-hybrid screen. In vascular endothelial cells, which only express -actinin-1 and -4, -actinin-4 interacted and colocalized with eNOS. Addition of -actinin-4 directly inhibited eNOS recombinant protein, and overexpression of -actinin-4 inhibited eNOS activity in eNOS-transfected COS-7 cells and bovine aortic endothelial cells (BAECs). In contrast, knockdown of -actinin-4 by siRNA increased eNOS activity in BAECs. The -actinin-4-binding site on eNOS was mapped to a central region comprising the calmodulin-binding domain, and the eNOS-binding site on -actinin-4 was mapped to the fourth spectrin-like rod domain, R4. Treatment of endothelial cells with a calcium ionophore, A23187, decreased -actinin-4-eNOS interaction, leading to translocation of -actinin-4 from plasma membrane to cytoplasm. Indeed, addition of calmodulin displaced -actinin-4 binding to eNOS and increased eNOS activity. These findings indicate that eNOS activity in vascular endothelial cells is tonically and dynamically regulated by competitive interaction with -actinin-4 and calmodulin.—Hiroi, Y., Guo, Z., Li, Y., Beggs, A. H., Liao, J. K. Dynamic regulation of endothelial NOS mediated by competitive interaction with -actinin-4 and calmodulin.