Maxi-K channels consist of a pore-forming α subunit and a regulatory β subunit, which confers the channel with a higher Ca²⁺ sensitivity. Estradiol bound to the β subunit and activated the Maxi-K channel (hSlo) only when both α and β subunits were present. This activation was independent of the generation of intracellular signals and could be triggered by estradiol conjugated to a membrane-impenetrable carrier protein. This study documents the direct interaction of a hormone with a voltage-gated channel subunit and provides the molecular mechanism for the modulation of vascular smooth muscle Maxi-K channels by estrogens.