Blockade of small-conductance Ca2+-activated K+ channels (SKCa) and intermediate conductance Ca2+- activated K+ channels (IKCa) can cause inhibition of endothelium-dependent hyperpolarizing factor (EDHF) in many vascular beds from animals, but there is a relative paucity of data in human vessels. Systemic arteries, isolated from women with healthy pregnancies, relax to the endothelial-dependent agonist bradykinin via a nonprostacyclin and non-nitric oxide pathway attributable to EDHF. Therefore, in this study, the authors investigated the effect of pharmacological blockade of SKCa and IKCa on EDHF-mediated relaxation of human omental and myometrial arteries preconstricted with either arginine vasopressin or U46619. Human arteries were isolated from omental and myometrial biopsies taken from healthy women undergoing planned cesarean section at term. Endothelial function was assessed using wire myography. In all vessels examined, nonspecific blockade of IKCa with charybdotoxin attenuated EDHF-attributed relaxation. However, when Tram 34 was used to block IKCa, the attenuation of relaxation was evident only with U46619 preconstriction. In arteries from both vascular beds, and with either preconstrictor, a combination of either apamin and charybdotoxin or apamin plus Tram 34 almost ablated EDHF-attributable relaxation. These data support the notion that in human systemic arteries, activation of, primarily, SKCa and IKCaK+ channel subtypes underlies EDHF-mediated relaxation. These results have important implications for future studies ascertaining the molecular mechanisms of hypertensive disorders (eg, preeclampsia, in which EDHF is thought to be aberrant). © 2007 by the Society for Gynecologic Investigation.