Endothelial cell stiffness, a crucial parameter of endothelial function, is increased by the mineralocorticoid hormone aldosterone. Aldosterone acting via the genomic pathway binds to intracellular mineralocorticoid receptors and activates epithelial sodium channels (ENaCs). We currently investigate whether aldosterone-induced ENaC activity significantly alters the membrane potential of bovine aortic endothelial cells (GM7373) and whether this membrane potential change could be responsible for the increase in cellular stiffness. To test these hypotheses we developed a novel experimental approach allowing the direct correlation of changes in membrane potential with changes in stiffness of an individual cell. Cellular stiffness can be determined with the atomic force microscope (AFM) recording force curves on the cell while the plasma membrane potential can be measured with fluorescence microscopy using voltage-sensitive dyes. For simultaneous stiffness- and membrane potential measurements we combined these two methods using a Bioscope II AFM integrated with a Leica DMI 6000 B inverted fluorescence microscope. With this method we could constantly (every 10 seconds) record force curves and at the same time fluorescence intensity of one and the same cell for a time span of 5 minutes. During this period of time and under the applied control conditions, both the stiffness- and the fluorescence intensity values remained virtually constant. The stiffness of the cells was 3.9 1.11 pN/nm and the membrane potential -67.4 8.15 mV. Depolarizing the cells to -2.8 mV using the sodium ionophore gramicidin resulted in a stiffness increase of 36%. In summary, we demonstrate a new method allowing simultaneous recordings of cellular mechanical properties and dynamic changes in intracellular fluorescence. We use this approach to study the relationship between membrane potential and stiffness in endothelial cells and to disclose the mechanisms underlying the change in endothelial cell stiffness mediated by aldosterone.