Features and kinetics of Adenovirus (Ad)-mediated gene transfer to endothelial cells (EC) are not ultimately determined. We tested variables pertinent to the efficiency of Ad-mediated gene transfer to bovine aortic endothelial cells (BAEC) including: (1) Ad-vectors with different promoters, (2) kinetics of transduction efficiency of LacZ gene to BAEC, (3) the concentration and volume of vector-containing medium, (4) the period of incubation time of Ad vectors with BAEC, (5) the duration of transgene expression. An Ad5-LacZ vector with a cytomegalovirus (CMV) promoter transduced the LacZ gene to the cells more efficiently than vectors with the Rous sarcoma virus (RSV) promoter. However, both vectors exhibited a dose-dependent relationship between the vector multiplicity of infection (moi) and the percentage of LacZ-expressing cells. The higher moi of both vectors achieved nearly 100% of transduction efficiency in cultured BAEC. Although the Ad-CMV-LacZ vector better transduced the LacZ gene to BAEC than Ad-RSV-LacZ, a long period of vector exposure to BAEC could overcome the slightly difference in transduction efficiency between the two vectors. These results indicate that both Ad vectors are efficient for gene transfer to endothelial cells, and higher moi of vectors or a longer period exposure of vectors to EC can facilitate efficient transduction of foreign gene into EC in culture.