Endothelium-dependent dilation is mediated by NO, prostacyclin, and an endothelium-derived hyperpolarizing factor (EDHF). EDHF-signalling is thought to be initiated by activation of endothelial Ca2+ -activated K+ channels (KCa ) leading to hyperpolarization of the endothelium and subsequently to hyperpolarization and relaxation of vascular smooth muscle. Here, we used a genetic approach to test the functional role of the endothelial intermediate-conductance KCa (KCa 3.1) in endothelial hyperpolarization, EDHF-mediated dilation, and in blood pressure control. Disruption of the KCa 3.1 gene in mice abolished endothelial KCa 3.1 currents. We detected no compensation by the other endothelial KCa channel KCa 2.3. As a consequence, endothelial and smooth muscle hyperpolarization in response to acetylcholine (ACh) was reduced in KCa 3.1-/- mice. EDHF-mediated dilations were greatly impaired in carotid arteries in KCa 3.1-/- mice. Moreover, the loss of KCa 3.1 led to a significant increase in arterial blood pressure and to mild left ventricular hypertrophy. These results indicate that the endothelial KCa 3.1 is a fundamental determinant of endothelial hyperpolarization, EDHF-signalling and thereby a crucial factor in the control of vascular tone and overall circulatory regulation.