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 alpha (slo1) subunit homotetramers, key biophysical and pharmacological properties are drastically modified by the presence of auxiliary beta subunits (encoded by KCNMB1-4). Numerous physiological steroids, including sex hormones, gluco- and mineralocorticoids, activate beta subunit-containing BK channels, yet these steroids appear to be sensed by different types of beta subunits, with some steroids being sensed by homomeric slo1 channels as well. We recently showed that beta1 sensitizes the BK channel to microM concentrations of lithocholate (LC). Following expression of rat cerebral artery myocyte slo1 subunits ("cbv1") with beta1, beta2, beta3 or beta4 in Xenopus laevis oocytes we now demonstrate that BK beta2, beta3 and beta4 subunits fail to substitute for beta1 in providing LC-sensitivity (150 microM) 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 beta1 is highly expressed, while failing to do so in skeletal (flexor digitorum brevis) muscle, where BK beta1 expression is negligible. This indicates that the native environment of the BK channel sustains the LC-sensitivity distinctly provided to the BK channel by beta1 subunits. Our study indicates that LC represents a unique tool to probe the presence of functional beta1-subunits and selectively activate BK channels in tissues that highly express KCNMB1.