Estrogen (17β-estradiol; 17βE) and xenoestrogens, estrogenic compounds that are not steroid hormones, have nongenomic actions at plasma membrane receptors unrelated to the nuclear estrogen receptor. The open probability (Po) (P.) of large conductance Ca2+/voltage-sensitive k+(BK) channels is increased by 17βE through the regulatory β1 subunit. The pharmacological nature of the putative membrane binding site is unclear. We probed the site by determining whether tamoxifen ((Z)-1-(p-dimethylaminoethoxy-phenyl)-1,2-diphenyl-1-butene; Tx), a chemotherapeutic xenoestrogen, increased Po in clinically relevant concentrations (0.1-10 μM). In whole cell patch clamp recordings on canine colonic myocytes, which express the β1 subunit, Tx activated charybdotoxin-sensitive K+ current. In single channel experiments, Tx increased the NPo (Po × number channels; N) and decreased the unitary conductance (γ) of BK channels. Tx increased NP o (EC50 = 0.65 μM) in excised membrane patches independent of Ca2+ changes. The Tx mechanism of action requires the β1 subunit, as Tx increased the NPo of Slo α expressed in human embryonic kidney cells only in the presence of the β1 subunit. Tx decreased γ of the α subunit expressed alone, without effect on NPo. Our data indicate that Tx increases BK channel activity in therapeutic concentrations and reveal novel pharmacological properties attributable to the α and β1 subunits. These data shed light on BK channel structure and function, non-genomic mechanisms of regulation, and physiologically and therapeutically relevant effects of xenoestrogens.