The endothelium derived relaxing factor nitric oxide (NO) plays a key role in blood pressure control. NO functions also as an important regulator of intracellular signalling pathways. In endothelial cells NO is produced by the calcium-dependent endothelial nitric oxide synthase (eNOS). Due to the variety of functions of NO, the regulation of eNOS is rather complex and the exact mechanisms are not yet completely understood. Recent experiments revealed a negative correlation between endothelial cell stiffness and NO release. A key hormone that influences cell stiffness as well as NO release is the mineralocorticoid aldosterone. In endothelial cells aldosterone acts via a fast non-genomic and a slow genomic pathway. Long-term exposure to aldosterone stiffens the cells and reduces NO production. During the rapid response to aldosterone (within the first 15 min), cells soften. The hypothesis was tested that rapid aldosterone-mediated cell softening is associated with NO release. Bovine aortic endothelial cells were treated with various concentrations of aldosterone (1 - 10 nM). Using NO selective electrodes, a tight correlation between aldosterone concentration and transient NO release was revealed. We also used a combined fluorescence and atomic force microscope to simultanously measure cell stiffness and NO release. To trace the NO inside the cell we loaded the cell with a fluorescent NO indicator. Again we detected a rapid aldosterone-induced NO burst in endothelial cells associated with a decrease in cell stiffness. Increased NO synthesis was inhibited in both experimental approaches by the eNOS-Blocker N-Nitro-L-arginine-methyl ester. Taken together, vascular endothelium responds to aldosterone with an instantaneous burst of NO followed by a long-term depression of NO production. The results support the view that cell stiffness and NO release are inversely correlated.