Cationic lipid-mediated gene delivery provides several key advantages over viral vectors for delivery of plasmid DNA into somatic cells, such as negligible immunogenicity, possibility of delivering larger DNA fragments, and ease of production and handling. However, the low transfection efficiency, as compared to their viral counterparts, remains an obstinate drawback that confronts the development of cationic lipoplexes to become a clinically-effective gene medicine. One specific problem is the lack of a meaningful structure-activity relationship for cationic lipids. Unfortunately, most of the work done so far in this aspect has been empirical, based mainly on the synthesis of cationic lipids and screening them for transfection activity. Other factors, such as the complexity of the lipofection process itself and its cell type dependency made the task even more challenging. Hence, the contribution of each part of the cationic lipid molecular architecture to the lipofection process is, as yet, inadequately understood. The current work is a part of a long term structure-activity relationship study utilizing dialkoylamidopropane-based cationic lipids for the delineation structural requirements of cationic lipids for high DNA transfection efficiencies. Specifically, the first aim of this project was the synthesis of 1,2-dialkoylamidopropane-based cationic lipid with varied acyl chain length, degree of unsaturation of the acyl chain and methyl substitution of the cationic polar head group of the lipid molecules. The second aim was the physicochemical characterization of these lipids and their assemblies in isolation as well as in the presence of helper lipids and plasmid DNA. The final aim was correlating the aforementioned physicochemical properties with the molecular architecture of the cationic lipids and their biological activity. To this end, two series of cationic lipids; N,N/-diacyl-1,2-diaminopropyl-3-carbamoyl-(dimethylaminoethane) and N,N/-diacyl-1,2-diaminopropyl-3-carbamoyl-(aminoethane) have been synthesized. The ability of these novel lipids to promote in vitro transfection activity was challenged against B16-F0 mouse melanoma cell line. In both series, only the dioleoyl derivatives, 1,2lmt[5] and 1,2lmp[5] mediated significant expression of the reporter gene. Maximum activity was obtained in the presence of the helper lipid DOPE at N/P molar ratio of 2. In agreement with the transfection results, characterization of the electrostatic interactions between these novel lipids and DNA indicated that the highly protonated dioleoyl species were most efficient in associating with DNA as compared to the saturated analogs. However, methyl substitution of the amine head groups appears to weaken this association but, nevertheless, it did not abolish in vitro transfection activity. The current findings suggest that efficient condensation of plasmid DNA by cationic lipids is not a requirement for improved in vitro transfection activity. Loose association between plasmid DNA and cationic lipids is enough to promote significant in vitro transfection activity. Several reports had found bilayer fluidity of monovalent cationic lipids to correlate with improved transfection activity. The results of the current work agree with these findings. Langmuir film balance and differential scanning calorimetry studies indicated that only the dioleoyl derivatives possessed the required fluidity and, thereby, mediated significant levels of in vitro reporter gene expression. Specifically, only monolayers of dioleoyl derivatives 1,2lmt[5] and 1,2lmp[5] exhibited a liquid-expanded phase state at all temperatures studied. Moreover, among the primary derivatives, only bilayers of 1,2lmp[5] derivative were found to exist in a liquid-crystalline state at 37 °C. In a novel approach using the Langmuir film balance technique, a correlation between in vitrotransfection activity of cationic lipids and their interfacial elasticity has been established. The transfection active dioleoyl analogs formed the most elastic monolayers as indicated by low compressibility moduli and slope values. Finally, a rule for DOPE in boosting transfection activity by promoting efficient association between DNA and cationic lipids is discussed.