Mono layers of the cationic lipid DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and cholesterol or heptafluorocholesterol were prepared, and their interaction with DNA was characterized. The mixture of DOTAP with each of the sterols at 1:1 molar ratios leads to monolayers in a liquid expanded state, similarly to that of DOTAP alone. The area per molecule of the mixtures was smaller than that expected according to the additivity rule applicable if the two components are either completely miscible or immiscible within the monolayer. The observed negative deviation from the additivity indicates the existence of additional attractive interactions between the components. The surface potential of DOTAP monolayer is positive (+560 mV). It decreases only slightly after the addition of cholesterol (+540 mV) but drastically after the addition of heptafluorocholesterol (+20 mV) in the 1:1 mixtures at a surface pressure of 35 mN/m. This difference is attributed to the negative dipole moment of the fluorinated component. The adsorption of DNA is similar for both systems, which supports the possibility of using fluorinated cholesterol as helper lipid in DNA transfection vectors.