Objective: The endothelial Ca2+-activated K+ channels IKCa (KCa3.1) and SKCa (KCa2.3) have been proposed to play an essential role in EDHF-mediated dilation by mediating endothelial and subsequent smooth muscle hyperpolarization. However, their in vivo roles in EDHF-signaling and the contribution of the IKCa/SKCa-EDHF system to blood pressure control have not been understood yet. Methods: To study the in vivo roles of endothelial IKCa and SKCa, we generated IKCa-deficient mice (KCa3.1-/-) and IKCa/SKCa-deficient mice (KCa3.1-/-/KCa2.3T/T+doxycycline (dox)), and studied endothelial KCa currents and hyperpolarization responses by patch-clamp and sharp electrode techniques, respectively, and determined EDHF-type-dilations and blood pressure alterations by using pressure myography and telemetry, respectively. Results: Single (KCa3.1-/- or KCa2.3T/T+dox) or combined IKCa/SKCa deficiency (KCa3.1-/-/KCa2.3T/T+dox) produced a significant reduction or loss of endothelial KCa currents, respectively. Smooth muscle hyperpolarization responses to acetylcholine were severely impaired by IKCa and combined IKCa/SKCa deficiency. The loss of IKCa alone and of both channels impaired acetylcholine-induced vasodilations, while the loss of SKCa abrogated EDHF-type dilations to shear stress stimulation. Hence, deficiency of either channel or of both produced an increase of blood pressure. This higher blood pressure was evident during spontaneous physical activity. Over-expression of SKCa in KCa3.1-/-/KCa2.3T/T-mice and pharmacological opening of IKCa in KCa2.3T/T+dox mice by injection of the novel IKCa-opener SKA-31 improved EDHF-type vasodilations and lowered blood pressure. Conclusion: Endothelial hyperpolarizing IKCa and SKCa (with distinct stimulus-dependent functions) are critical molecular determinants in EDHF-type vasodilation and blood pressure control in vivo. Therefore, pharmacological targeting of these ion channels may offer new ways to treat hypertension and cardiovascular disease associated with endothelial dysfunction.