The β configuration of the sterol C3 hydroxyl is crucial for cholesterol and related monohydroxysterols to inhibit BK channels. Representative single-channel recordings obtained after cbv1 incorporation into control (A), epicholesterol- (B), epicoprostanol- (C), or epicholestanol-containing (D) POPE/POPS (3:1 wt/wt) bilayers. Sterols were introduced at 33 mol% in the lipid mixture. For A–D, channel openings are shown as upward deflections; arrows on the top trace of each panel indicate the baseline. Po was calculated as described in the legend to Fig. 2. (E) Scatter graph showing individual Po values in control and in the presence of epicholesterol, epicoprostanol, or epicholestanol in the lipid mixture. Each data point corresponds to one independent bilayer preparation. (F) Averaged Po values show that epicholesterol, epicoprostanol, and epicholestanol failed to reduce BK channel activity from control values. (G) Averaged degree of sterol-induced modification of cbv1 channel displayed as a percentage of inhibition from control bilayers. Data from cholesterol-containing bilayers are shown as positive control. **, significantly different from controls (P < 0.01).