Clusters of positively-charged basic amino acid residues, particularly lysine, are known to promote the interaction of many peripheral membrane proteins with the cytoplasmic surface of the plasma membrane via electrostatic interactions. In this work, cholesterol's effects on the interaction between lysine residues and membranes have been studied. Using poly-l-lysine (PLL) and vesicles as models to mimic the interaction between lysine-rich protein domains and the plasma membrane, light scattering measurements indicated cholesterol enhanced the electrostatic interaction through indirectly affecting the negatively charged phospholipid dioleoylphosphatidylserine, DOPS. Addition of PLL to lipid vesicles containing DOPS showed an initial increase in static light scattering (SLS), attributed to binding of PLL to the vesicle surface, followed by a slower continuously declining SLS signal, which, from comparison with fluorescent dye leakage studies could be attributed to vesicle lysis. Although electrostatic interactions between PLL and the membrane were not necessary for penetration to occur, cholesterol promoted membrane disruption of negatively charged vesicles, possibly by increasing the electrostatic interactions between PLL and the membrane. In contrast, cholesterol lowered the susceptibility of uncharged vesicles (formed using dioleoylphosphatidylcholine, DOPC) to PLL penetration. This can be explained by the absence of electrostatic interactions and cholesterol's known ability to increase membrane thickness and mechanical strength. Thus, the ability of cationic peptides to penetrate membranes including cholesterol is likely to depend on the membrane's PS:PC ratio.