Large conductance, calcium- and voltage-gated potassium (BK) channels regulate numerous physiological processes. While most basic functional characteristics of native BK channels are reproduced by BK α (slo1) subunit homotetramers, key biophysical and pharmacological properties are drastically modified by the presence of auxiliary β subunits (encoded by KCNMB1–4). Numerous physiological steroids, including sex hormones, gluco- and mineralocorticoids, activate β subunit-containing BK channels, yet these steroids appear to be sensed by different types of β subunits, with some steroids being sensed by homomeric slo1 channels as well. We recently showed that β1 sensitizes the BK channel to μM concentrations of lithocholate (LC). Following expression of rat cerebral artery myocyte slo1 subunits (“cbv1”) with β1, β2, β3 or β4 in Xenopus laevis oocytes we now demonstrate that BK β2, β3 and β4 subunits fail to substitute for β1 in providing LC-sensitivity (150μM) to the BK channel. These findings document for the first time a rather selective steroid activation of BK channels via a particular channel accessory subunit. In addition, LC routinely activated native BK channels in myocytes freshly isolated from rat cerebral artery smooth muscle, where BK β1 is highly expressed, while failing to do so in skeletal (flexor digitorum brevis) muscle, where BK β1 expression is negligible. This indicates that the native environment of the BK channel sustains the LC-sensitivity distinctly provided to the BK channel by β1 subunits. Our study indicates that LC represents a unique tool to probe the presence of functional β1-subunits and selectively activate BK channels in tissues that highly express KCNMB1.