Here we describe the first synthesis-screening approach for the identification and optimization of new cationic lipids for gene transfer in various cell lines. Combinatorial solid-phase chemistry was used to synthesize a library of new cationic lipids based on 3-methylamino-1,2-dihydroxypropane as the polar, cationic lipid part. As the nonpolar lipid part, different hydrocarbon chains were bound to the amino group of the scaffold and the amino group was further methylated to afford constantly cationic lipids. Lipids were synthesized in both configurations and as racemates, and the counter ions were also varied. By using a fully automated transfection screening method and COS-7 cells, the cationic lipid N,N-ditetradecyl-N-methyl-amino-2,3-propanediol (KL-1-14) was identified as a candidate lipid for the development of an improved transfection reagent. Screening the transfection properties of KL-1-14 in numerous combinations with the helper lipids dioleoylphosphatidylethanolamine (DOPE) and cholesterol (Chol) revealed that Chol is the most suitable helper lipid and the best KL-1-14/Chol ratio is 0.5-0.7. Compared to the standard transfection lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), transfection efficiency was improved by a factor of about 40. Furthermore, by using R- and S-configured KL-1-14, it could be shown that the configuration of the lipids had no significant influence on its transfection efficiency. The highest transfection efficiencies were achieved with chloride as the counter ion. The new lipofection reagent was further tested to transfect the cell lines MDA-MB-468, MCF-7, MDCK-C7, and primary dentritic cells (DC), which are important for the development of new anticancer gene therapy strategies. Even in these cells, KL-1-14/Chol (1:0.6) had improved transfection efficiencies, which were about two to four times higher than for DOTAP.