We examined the effects of the cannabinoid anandamide (AEA) and its stable analogue, methanandamide (methAEA), on large-conductance, Ca²⁺-activated K⁺ (BK) channels using HEK293 cells, in which the -subunit of the BK channel (BK), both and 1 (BK1), or both and 4(BK4) subunits were heterologously expressed. In a whole-cell voltage-clamp configuration, each cannabinoid activated BK in a similar concentration range. Because methAEA could potentiate BK, BK1, and BK4 with similar efficacy, the subunits may not be involved in the site of action for cannabinoids. Under cell-attached patch-clamp conditions, application of methAEA to the bathing solution increased BK channel activity; however, methAEA did not alter channel activity in the excised inside-out patch mode even when ATP was present on the cytoplasmic side of the membrane. Application of methAEA to HEKBK and HEKBK did not change intracellular Ca²⁺ concentration. Moreover, methAEA-induced potentiation of BK channel currents was not affected by pretreatment with a CB1 antagonist (AM251), modulators of G proteins (cholera and pertussis toxins), or application of a selective CB2 agonist (JWH133). Inhibitors of calmodulin, protein kinase G, and MAP kinases (W7, KT5823, and PD98059) did not affect the potentiation. Application of methAEA to mouse aortic myocytes significantly increased BK channel currents. This study provides the first direct evidence that unknown factor(s) in the cytoplasm mediate the ability of endogenous cannabinoids to activate BK channel currents. Cannabinoids may be hyperpolarizing factors in cells, such as arterial myocytes, where BK channels are highly expressed.