Retroviral vector-mediated gene transfer into endothelial cells is relatively inefficient with transduction rates as low as 1–2% in vitro and even lower in vivo. To increase the efficiency of gene transfer into endothelial cells, we used retroviral vectors expressing β-galactosidase and urokinase and measured endothelial cell transduction efficiencies with quantitative assays for β-galactosidase and urokinase protein. We evaluated several techniques reported to improve the efficiency of retroviral transduction in vitro, including 1) extended periods of exposure to vector, 2) repeated exposures to vector, 3) maximization of the ratio of vector particles to endothelial cells by increasing the volume and concentration of vector particles or by decreasing the number of endothelial cells exposed, 4) cocultivation of endothelial cells with vector-producing cells, and 5) variation of the type and concentration of polycation used with the retroviral vector. Only the use of more concentrated (higher titer) vector-containing supernatant and the use of the polycation DEAE-dextran improved the efficiency of gene transfer into endothelial cells in vitro. In an optimized transduction protocol, a 60-second exposure to 1 mg/ml DEAE-dextran followed by a single 6-hour exposure to supernatant of a titer of 105–106colony-forming units/ml resulted in transduction efficiencies of 50–90% with both vectors. Decreasing the time of the supernatant exposure to 15 minutes permitted transduction efficiencies of 15–20% while significantly minimizing the duration of the transduction. Therefore, the optimized protocol allows high efficiency in vitro gene transfer into endothelial cells within several hours. The briefer protocol may prove useful for in vivo gene transfer in which the time of exposure to the supernatant is limited.