Recently developed transfection methods for mammalian cells provide a powerful means for the study of gene function. Unfortunately, human endothelial cells were relative refractory to the classic transfection techniques. In this study we compared the usability of calcium phosphate, DEAE-dextran transfection, transferrinfection, lipofection, and electroporation for the transfection of early passage HUVECs and for the human endothelial cell lines ECV 304 and EA.hy 926. The classic transfection methods resulted in no or only marginal expression of the reporter gene E. coli beta-galactosidase. For lipofection experiments we compared the commercially available liposome formulations DOTAP and Transfectam with liposomes prepared of dimethyldioctadecylammoniumbromide (DDAB) or 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide (DMRIE) as the cationic lipid compound and dioleylphosphatidylethanolamine (DOPE) or Azolectin (a crude fraction of soybean lipids, commercially available as phosphatidylcholine II) as neutral co-lipid. Because the protocol for the chemical synthesis of DMRIE has not been published yet, we developed a protocol for the chemical synthesis of this cationic lipid. With transfection protocols optimized for each cell line, we could achieve transfection efficiencies up to 2%. Compared to the other methods used, the lipofection proved to be a reliable technique for the efficient transfection of the human endothelial cell lines ECV 304 and EA.hy 926. Although we achieved a maximum transfection efficiency of 0.45% for the lipofection of HUVEC, the electroporation seemed to be the better choice for these cells.