Endocannabinoid anandamide induces endothelium-dependent relaxation commonly attributed to stimulation of the G-protein coupled endothelial anandamide receptor. The study addressed the receptor-independent effect of anandamide on large conductance Ca2+-dependent K+ channels expressed in endothelial cell line EA.hy926. Under resting conditions, 10 μM anandamide did not significantly influence the resting membrane potential. In a Ca2+-free solution the cells were depolarized by ˜10 mV. Further administration of 10 μM anandamide hyperpolarized the cells by ˜8 mV. In voltage-clamp mode, anandamide elicited the outwardly rectifying whole-cell current sensitive to paxilline but insensitive to GDPβS, a G-protein inhibitor. Administration of 70 μM Mn2+, an agent used to promote integrin clustering, reversibly stimulated whole-cell current, but failed to further facilitate the anandamide-stimulated current. In an inside-out configuration, anandamide (0.1–30 μM) facilitated single BKCa channel activity in a concentration-dependent manner within a physiological Ca2+ range and a wide range of voltages, mainly by reducing mean closed time. The effect is essentially eliminated following chelation of Ca2+ from the cytosolic face and pre-exposure to cholesterol-reducing agent methyl-β-cyclodextrin. O-1918 (3 μM), a cannabidiol analog used as a selective antagonist of endothelial anandamide receptor, reduced BKCa channel activity in inside-out patches. These results do not support the existence of endothelial cannabinoid receptor and indicate that anandamide acts as a direct BKCa opener. The action does not require cell integrity or integrins and is caused by direct modification of BKCa channel activity.