We demonstrated previously that BK (K(Ca)1.1) channel activity (NP(o)) increases in response to bisphenol A (BPA). Moreover, BK channels containing regulatory β1 subunits were more sensitive to the stimulatory effect of BPA. How BPA increases BK channel NPo remains mostly unknown. Estradiol activates BK channels by binding to an extracellular site, but neither the existence nor location of a BPA binding site has been demonstrated. We tested the hypothesis that an extracellular binding site is responsible for activation of BK channels by BPA. We synthesized membrane-impermeant BPA-monosulfate (BPA-MS) and used patch clamp electrophysiology to study channels composed of α or α + β1 subunits in cell-attached (C-A), whole-cell (W-C), and inside-out (I-O) patches. In C-A patches, bath application of BPA-MS (100 μM) had no effect on the NP(o) of BK channels, regardless of their subunit composition. Importantly, however, subsequent addition of membrane-permeant BPA (100 μM) increased the NP(o) of both α and α + β1 channels in C-A patches. The C-A data indicate that in order to alter BK channel NP(o), BPA must interact with the channel itself (or some closely associated partner) and diffusible messengers are not involved. In W-C patches, 100 μM BPA-MS activated current in cells expressingα subunits, whereas cells expressing α + β1 subunits responded similarly to a log-order lower concentration (10 μM). The W-C data suggest that an extracellular activation site exists, but do not eliminate the possibility that an intracellular site may also be present. In I-O patches, where the cytoplasmic face was exposed to the bath, BPA-MS had no effect on the NP(o) of BK α subunits, but BPA increased it. BPA-MS increased the NP(o) of α + β1 channels in I-O patches, but not as much as BPA. We conclude that BPA activates BK α via an extracellular site and that BPA-sensitivity is increased by the β1 subunit, which may also constitute part of an intracellular binding site.