Modulations in the plasticity (stiffness) of the endothelial cell cortex (i.e. a 100 nm zone underneath the plasma membrane) directly influence the release of the main endothelial vasodilator nitric oxide (NO). A decrease in cortical stiffness enhances the activity of the endothelial NO synthase (eNOS). The exact mechanism of stiffness-dependent eNOS activation, however, is unknown. We hypothesize that cortical stiffness modulations are due to dynamic changes in the submembranous actin web. A depolymerization (=softening) leads to an increased interaction of globular actin (G-actin) and eNOS. This interaction, in turn, fosters endothelial NO release. To test this hypothesis, we applied simultaneous atomic force (AFM) and fluorescence microscopy. The AFM-based nanoindentation technique allows an examination of nanomechanical properties of living cells. Detection of actin and NO by fluorescent Lifeact-eGFP and DAF-FM/DA was used to define the polymerization status of cortical actin and the amount of NO release, respectively. By the application of the specific inhibitor for eNOS/actin interaction (P326TAT), we show that the submembranous G-actin associates with eNOS, thus increasing NO release. This elevation of eNOS activity strictly depends upon cortical actin dynamics and does not depend upon actin dynamics in the bulk phase of the cell. We conclude that cortical actin dynamics regulate eNOS and thus control vascular tone and blood pressure.