Nitric oxide (NO) is a crucial endothelium-derived relaxing factor in the vascular system. It is a small gaseous uncharged free radical containing one unpaired electron. In endothelial cells NO is synthesized by the Ca²⁺-dependent nitric oxide synthase (eNOS). Since NO is a very unstable molecule and decomposes to NO metabolites soon after its generation (half life time: ~0.5 to 5 s), the range of its activity is obviously limited. Therefore, it is difficult to definitely show the impact of NO "per se" because the effects of NO metabolites (NO₂, NO₂^-...) cannot be excluded. NO synthesis is a highly regulated process and still unclear. Recent experiments indicate that NO release is influenced by the mechanical stiffness of endothelial cells. Modulation of cell stiffness by disruption of the cortical cytoskeleton induces an increase of NO production. Mediators like the mineralcorticoid hormone aldosterone also influence the NO release by modifying cell stiffness. However, a direct proof of changes in NO production as a result of changes in cell stiffness is still lacking. The only way to directly measure changes of NO immediately after synthesis is amperometry using NO-selective electrodes. We are now using nafion-coated carbon fibre electrodes which are small in size (7 mm), highly selective for NO and show a low detection limit (~0.5 nM). Using these electrodes we can observe in human umbilical venous endothelial cells (HUVEC) at the single cell level dynamic changes of NO release at a high temporal resolution. Using the Ca²⁺ ionophore ionomycin we induce maximal activation of eNOS and measure transient NO concentration changes of 16.4 3.6 nmol/l (n=8), with the electrode tip located in close vicinity to the apical cell membrane. By treatment with aldosterone cell stiffness can be changed and rapid NO release can be analyzed by using the ionomycin-peak as the gold standard for quantification.