Cationic liposome transfection reagents are useful for transferring polynucleotides into cells, and have been proposed for human pulmonary gene therapy. The effect of adding cholesterol to cationic lipid preparations has been tested by first formulating the cationic lipid N-[1-(2,3-dioleoyloxy)propyl-N-[1-(2-hydroxy)ethyl]-N,N-dimethyl ammonium iodide (DORI) with varying amounts of dioleoylphosphatidylethanolamine (DOPE) and cholesterol. Cholesterol was found to enhance lipid-mediated transfection in both the respiratory epithelial cells and mouse fibroblasts. These findings will facilitate nucleic acid transfection of many cell types including differentiated epithelial cell monolayers, and therefore may be useful for examining gene regulation in various cell types and for developing pulmonary gene therapy.