Ca²⁺-activated K⁺ channels (K_Ca) are important regulators of endothelial function by mediating endothelial hyperpolarization and thus endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilation. In addition the cytochrome P450 (CYP) pathway has also been proposed to contribute to EDHF signalling by intraendothelial mechanisms or by the release of epoxyeicosatrienoic acids (EETs). Here we evaluated whether the endothelial K_Ca subtype K_Ca3.1 is modulated by the CYP pathway. In isolated aortic endothelial cells, the preactivated K_Ca3.1 current (cell dialysis with 0.5 mM calcium) was twofold enhanced by the subsequent application of 1 mM 5,6-EET or 11,12-EET. In contrast, the precursor of EETs, arachidonic acid (AA) blocked K_Ca3.1 currents. In conclusion, the potentiating effect of EETs on K_Ca3.1 activity may represent a novel intraendothelial mechanism by which the CYP pathway may contribute to EDHF signalling. Since AA blocks the K_Ca3.1 activity, the CYP-mediated metabolism of AA may be required to unblock the K_Ca3.1. This might be important for enabling adequate endothelial hyperpolarization and thus EDHF-mediated vasodilation.