Studies of the contribution of various physical properties of cationic lipid/DNA complexes (CLDCs) to their observed transgene expression in vitrowere conducted using cationic liposomes composed of the cationic lipids 1,2‐dioleoyl‐3‐trimethylammonium propane (DOTAP) and dimethyldioctadecylammonium bromide (DDAB), with or without equimolar amounts of cholesterol (CHOL) or 1,2‐dioleoylphosphatidylethanolamine (DOPE). The relative degree of luciferase expression by CLDCs is dependent on a complex relationship between net charge of the CLDC as well as previously reported properties, such as membrane fluidity and curvature of the cationic bilayer. Assessments were made of the role of these physical properties on CLDC stability in the extracellular medium, the extent of DNA cellular association, and membrane disruption activity. The efficiency of luciferase expression from negatively charged CLDCs is greatly improved by incorporation of DOPE. This result correlates with enhanced resistance to inhibition of gene delivery by heparan sulfate, increased cellular association of DNA, and enhanced membrane disruption activity. Luciferase expression by positively charged CLDCs is greatly reduced by incorporating equimolar amounts of CHOL and DOPE. This result occurs is in spite of increased resistance to heparan sulfate‐mediated inhibition of gene delivery, increased DNA cellular association, and enhanced membrane disruption activity. The observed CLDC compositional effects on luciferase expression along with observed effects on the delivery process suggest that a better understanding of the kinetics and specific routes of gene delivery is necessary. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:108–123, 2004