Endothelial dysfunction is frequently involved in the pathogenesis of vascular disease. While nitric oxide (NO) inhibits smooth muscle cell proliferation, its effect on endothelial cell proliferation is unclear. The aim of this study was to determine if adenoviral-mediated gene transfer of endothelial NO synthase (eNOS) to human umbilical vein endothelial cells (HUVECs) would result in increased generation of NO and affect endothelial cell proliferation. HUVECs were transduced with adenoviral vectors encoding eNOS (AdeNOS) or β-galactosidase (Adβgal) or exposed to diluent (control). AdeNOS-transduced cells showed increased eNOS expression as detected by Western blot analysis, and increased concentrations of cGMP (control 0.7 ± 0.1; Adβgal 0.9 ± 0.2; AdeNOS 3.1 ± 0.5 pmol/mg protein; p < 0.001) and nitrite (control 11.8 ± 1.2; Adβgal 13.3 ± 1.7; AdeNOS 21.1 ± 2.2 nmol/mg protein/hour; p < 0.01). DNA synthesis as assessed by [<sup>3</sup>H]thymidine incorporation and cell counts were significantly reduced (by ∼30%) in AdeNOS-transduced HUVECs. Expression of mitogen-activated protein kinase was also decreased in AdeNOS-transduced cells. This study shows that adenoviral-mediated gene transfer of eNOS to HUVECs inhibits endothelial cell proliferation.