Smooth-muscle calcium-activated large-conductance potassium channels (BK channels) are activated by tamoxifen and 17-β-estradiol. This increase in NPo, the number of channels, N, multiplied by open probability, depends on the presence of the regulatory β1-subunit. Furthermore, a previous study indicated that 17-β-estradiol might bind an extracellular site on the β1-subunit. Because tamoxifen and 17-β-estradiol may share a common binding site, we hypothesized that tamoxifen activates BK channels through a site on the extracellular surface of the membrane. A membrane-impermeant analog of tamoxifen, ethylbromide tamoxifen, was synthesized and used to test this hypothesis in whole-cell, outside-out, cell-attached, and inside-out patches from canine colonic smooth muscle cells. Ethylbromide tamoxifen is positively charged and is therefore membrane-impermeant. In whole-cell experiments, ethylbromide tamoxifen increased K+ current at potentials positive to +40 mV, which has previously been attributed to BK channels. Unlike tamoxifen, ethylbromide tamoxifen did not inhibit delayed rectifier current. In outside-out patches, ethylbromide tamoxifen increased BK channel NPo with an EC50 value of 1 μM. Ethylbromide tamoxifen did not increase BK channel NPo in cell-attached or inside-out patches; however, subsequent addition of equimolar tamoxifen did. Both drugs diminished BK channel unitary conductance to a degree that paralleled the effect on NPo, suggesting an additional interaction with the pore-forming a-subunit. An interaction of tamoxifen with the pore was supported by a right shift in the concentration-response curve for tetraethylammonium; similar results were evident with iberiotoxin and charybdotoxin block. Our data suggest that ethylbromide tamoxifen does not easily traverse the plasma membrane and that tamoxifen binding responsible for activation of BK channels is at an extracellular site. The tamoxifen binding site may be within the extracellular loop of the BK channel β1-subunit or, alternatively, on an as-yet-unidentified mediator that has an extracellular binding site.