Cationic lipids (cytofectins) have gained widespread acceptance as pharmaceutical polynucleotide delivery agents for both cultured cell and in vivo transfection, and the cytofectins DOTAP and DC-Cholesterol are being tested in clinical human gene therapy trials. This study reports the effects of modifications in the hydrophobic domain of a prototypic cytofectin (DORI), including modifications in lipid side-chain length, saturation, and symmetry. A panel of related compounds was prepared and analyzed using DNA transfection, electron microscopy, and differential scanning calorimetry (DSC). Lipid formulations were prepared with dioleoylphosphatidylethanolamine (DOPE) as unsonicated preparations and sonicated preparations. Transfection analyses were performed using cultured fibroblasts, human bronchial epithelial, and Chinese hamster ovarian cells as well as a mouse model for pulmonary gene delivery. In general, cytofectins containing dissymmetric hydrophobic domains were found to work as well or better than the best symmetric analogs. Optimal side-chain length and symmetry varied with cell type. Compounds with phase transitions (Tc) above and below physiological temperature (37 degrees C) were tested for DNA transfection activity. In contrast to previous reports, cytofectin Tc was not found to be predictive of transfection efficacy. Pulmonary treatment with free DNA was found to be at least as effective as treatment with commonly used cytofectin:DNA complexes. However, cytofectins that incorporate a hydroxyethylammonium moiety in the polar domain were found to enhance in vivo gene delivery relative to free DNA.