Vascular actions of steroid hormones have gained increasing importance. Indeed, some steroid hormones favorably influence vascular structure and function, whereas others are detrimental. This review will focus on the endothelial effects of steroid hormones. In the first part, we summarize data from in vivo studies elucidating the regulation of endothelial function by steroid hormones. Accumulating data argue for an improvement of endothelium-derived relaxation and impaired vascular contraction by estradiol, whereas testosterone, progesterone, and aldosterone have contrary effects. In the second part, we present data from novel atomic force microscopy studies performed in living endothelial cells under the influence of steroid hormones. These studies provide insight into structural and functional alterations of endothelial cells characterized by changes in volume, apical surface, and stiffness. We summarize the available evidence that changes in shape of endothelial cells translate into changes of endothelial cell stiffness. Under the influence of estradiol, endothelial cells become spherical with consecutive improvement of elasticity, whereas aldosterone flattens endothelial cell-shape leading to increased stiffness. Both, endothelial cell shape and stiffness are major determinants of endothelial nitric oxide production. These studies emphasize the great potential of atomic force microscopy to investigate the function of living endothelial cells.