The endothelium is now recognized not only as a physical barrier between blood and vascular wall, but also as an important and strategically located organ with multiple endocrine and paracrine functions. By releasing vasoactive substances, the endothelium acts as an inhibitory regulator of vascular contraction, leukocyte adhesion, vascular smooth muscle cell growth, and platelet aggregation. This review intends to demonstrate how much the picture of the biological functions of nitric oxide has changed in cardiovascular physiology, extending beyond its vessel-relaxing activity, as well as to highlight new insights into the factors affecting its bioavailability and regulation in relation with many cardiovascular diseases.