Lipofection of nondividing cells is not efficient due to poor endosome escape and poor nuclear import. In vivo, the low division rate of endothelium makes them a difficult target for nonviral gene transfer. Using a cytoplasmic transcription assay by lipofecting pT7bgal with T7 RNA polymerase, we observed that nearly 80 % of confluent bovine aortic endothelium expressed lipofected plasmid, indicating that endosome escape was widespread in a nondividing cell population, albeit inefficient since most of the plasmid was observed in an endosome compartment. We then tested a classical nuclear localization sequences (NLS) to understand its role on plasmid expression during lipofection. We utilized the NLS of the SV40 T antigen (CGYGPKKKRKVGG). Increasing amounts (2 to 80 μg) of SV40 T-antigen were complexed with pCMVb (2 μg) and delivered to dividing subconfluent BAEC with lipofectamine. We found that the SV40 T-antigen sequence caused a dose-dependent enhancement of transfection efficiency in BAEC from about 2 to 10 % (n=3). However, the transfection efficiency was below 10% suggesting that only the dividing cells in the monolayer were susceptible to lipofection. Similar effects were seen with a scrambled sequence of SV40 T-antigen NLS or lipofection with replication-deficient adenovirus (which enhanced endosome escape by 117-fold). Toward overcoming the final rate limit of nuclear import during nonviral gene transfer, we conjugated a nonclassical NLS, the M9 sequence of hnRNP A1, to a cationic peptide. Lipofection of highly confluent BAEC with plasmid conjugated with the conjugate peptide resulted in 83% transfection and a 63-fold increase in marker gene expression. The M9-cationic peptide localized fluorescent plasmid into the nucleus of permeabilized cells, and this activity was inhibited by wheat germ agglutinin. Plasmid alone was not imported into the nucleus in this assay. Combining the M9 sequence with DNA binding activity to enhance gene transfer may eventually lead to viral and lipid free proteofection of nondividing cells.