Liposome-mediated transfection of endothelial cells provides a valuable experimental technique to study cellular gene expression and may also be adapted for gene therapy studies. However, the widely recognized disadvantage of liposome-mediated transfection is low efficiency. Therefore, studies were performed to optimize transfection techniques in human endothelial cells. The majority of the experiments were performed with primary cultures of human umbilical vein endothelial cells (HUVEC). In addition, selected experiments were performed using human brain microvascular endothelial cells and human dermal microvascular endothelial cells. To study transfection rates, HUVEC were transfected with the pGL3 vector, containing the luciferase reporter gene, complexed with several currently available liposomes, such as different Perfect Lipid (pFx) mixtures, DMRIE-C, or lipofectin. The optimal transfection rate was achieved in HUVEC transfected for 1.5 h with 5 µg/ml of DNA plasmid in the presence of 36 µg/ml of pFx-7. In addition, transfection with the VR-3301 vector encoding for human placental alkaline phosphatase revealed that, under the described conditions, transfection efficiency in HUVEC was approximately 32%. Transfections mediated by other liposomes were less efficient. The usefulness of the optimized transfection technique was confirmed in HUVEC transfected with NF-ĸB or AP-1-responsive constructs and stimulated with TNF or LPS. We conclude that among several currently available liposomes, pFx-7 appears to be the most suitable for transfections of cultured human endothelial cells.