The contribution of small (SK_Ca) and intermediate (IK_Ca) conductance, Ca²⁺-activated K⁺ channels to nitric oxide (NO) generation by Ca²⁺-mobilizing stimuli was investigated in human umbilical vein endothelial cells (HUVECs) by combining DAF-FM and Fluo-3 microfluorimetry with perforated patch clamp recordings to monitor stimulated NO synthesis, cytosolic Ca²⁺ transients and membrane hyperpolarization. ATP or histamine evoked reproducible elevations in NO synthesis and cytosolic Ca²⁺ that were tightly associated with membrane hyperpolarizations. Whereas evoked NO synthesis was unaffected by either tetraethylammonium or BaCl₂ + ouabain, the SERCA pump inhibitor thapsigargin or removing external Ca²⁺ inhibited NO production, as did exposure to 80 mmol/L external KCl. Importantly, apamin and charybdotoxin (ChTx)/TRAM-34, selective blockers SK_Ca and IK_Ca channels, respectively, abolished both stimulated NO synthesis and membrane hyperpolarization and decreased evoked Ca²⁺ transients. They also inhibited an agonist-induced outwardly rectifying current characteristic of SK_Ca and IK_Ca channels. Under voltage clamp control, we further observed that the magnitude of agonist-induced NO production varied directly with the degree of membrane hyperpolarization. Mechanistically, our data indicate that SK_Ca and IK_Ca channel-mediated hyperpolarization represents a critical early event in agonist-evoked NO production by regulating the influx of Ca²⁺ responsible for eNOS activation. Moreover, it appears that the primary role of agonist-induced release of intracellular Ca²⁺ stores is to trigger the opening of both K_Ca channels, along with Ca²⁺ entry channels at the plasma membrane. Finally, the observed inhibitory effects of apamin and ChTx/TRAM-34 demonstrate that SK_Ca and IK_Ca channels are essential for NO-mediated vasorelaxation.