Biological membranes are heterogeneous structures with complex electrostatic profiles arising from lipids, sterols, membrane proteins, and water molecules. We investigated the effect of cholesterol and its derivative 6-ketocholestanol (6-kc) on membrane electrostatics by directly measuring the dipole electric field ((F) over right arrow (d)) within lipid bilayers containing cholesterol or 6-kc at concentrations of 0-40 mol% through the vibrational Stark effect (VSE). We found that adding low concentrations of cholesterol, up to similar to 10 mol %, increases (F) over right arrow (d), while adding more cholesterol up to 40 mol% lowers (F) over right arrow (d). In contrast, we measured a monotonic increase in (F) over right arrow (d) as 6-kc concentration increased. We propose that this membrane electric field is affected by multiple factors: the polarity of the sterol molecules, the reorientation of the phospholipid dipole due to sterol, and the impact of the sterol on hydrogen bonding with surface water. We used molecular dynamics simulations to examine the distribution of phospholipids, sterol, and helix in bilayers containing these sterols. At low concentrations, we observed clustering of sterols near the vibrational probe whereas at high concentrations, we observed spatial correlation between the positions of the sterol molecules. This work demonstrates how a one-atom difference in a sterol changes the physicochemical and electric field properties of the bilayer.