This paper documents the discovery that simple quaternary ammonium-based surfactants can recognize cholesterol-rich phospholipid membranes and that proton-ionizable analogs cannot. Specifically, hexadecyltrimethylammonium bromide (1a) and 3-(hexadecyldimethylammonio)propane-1-sulfonate (1b) have been shown to be effective in disrupting cholesterol-poor but not cholesterol-rich bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). In striking contract, hexadecyldimethylamine hydrogen bromide (1c) and hexadecylethylmethylamine hydrogen bromide (1d) were found to be very active against both types of targets. These findings reveal major differences between membrane-disrupting surfactants that bear a fixed positive charge and those that can eliminate such charge via deprotonation; they also indicate that repulsive forces between pendant ammonium groups and the hydrophobic interior of lipid bilayers can be used to modulate membrane disruption. The implications of these findings for the rational design of membrane-disrupting drugs are briefly discussed.