Endothelial cells have complex roles in the pathophysiology of vascular and heart disease and are increasingly being recognized as targets for gene therapy. The intravenous administration of plasmid DNA complexed to lipid tends to target transfection of endothelial cells within the lung; however, expression from the transgene remains transient. Here we utilize the integrating capability of the Sleeping Beauty (SB) transposon for durable gene transfer within lung endothelia. To restrict expression of the transgene, an endothelial cell-specific promoter, endothelin-1, was placed within the transposon. Further refinements to the transposon increased in vitro transposition efficiency by 3.6-fold. Utilizing this optimized transposon we evaluated the expression of two reporter molecules, secreted alkaline phosphatase (SEAP) and intracellular GFP, following administration of DNA-polyethylenimine complexes to mice. Long-term expression (>2 months) of SEAP occurred only with cotransfection of adequate amounts of transposase. Localization studies using the GFP reporter, at 3 days and 6 weeks postinjection, demonstrated that the majority of transgene-expressing cells were of endothelial origin, while the second most abundant cell type was type II pneumocyte. These results suggest that the SB transposon can be adapted to target particular cell types, in this case, endothelial cells. Such an approach may be useful for gene therapy paradigms involving the long-term modulation of vascular and endothelial cell biology.