—Gene transfer with replication-deficient adenovirus is a useful tool to study vascular biology. We have reported that overexpression of endothelial nitric oxide (NO) in carotid arteries from normal rabbits augments vasorelaxation mediated by NO. In this study, we tested the hypothesis that adenovirus-mediated gene transfer of endothelial nitric oxide synthase (eNOS) improves impaired relaxation of atherosclerotic vessels. We used 2 replication-deficient adenoviruses: AdeNOS, which carries cDNA for eNOS, and Adβgal, which expresses β-galactosidase. Common carotid arteries from 10 New Zealand White (NZW; plasma cholesterol, 79±13 mg/dL) and 10 Watanabe heritable hyperlipidemic (WHHL; plasma cholesterol, 452±39 mg/dL) rabbits were incubated in organ culture with AdeNOS, Adβgal, or vehicle alone. Carotid arteries from WHHL rabbits had mild to moderate atherosclerotic lesions. Histochemical staining for β-galactosidase and immunohistochemistry for eNOS indicated transgene expression in the endothelium and adventitia in both NZW and WHHL rabbits. Expression of eNOS determined with Western blot analysis after incubation with AdeNOS tended to be higher in vessels from WHHL rabbits than NZW rabbits. Effects of transgene expression on vascular function were examined by recording isometric tension 1 day after transduction. After precontraction with phenylephrine, acetylcholine produced significantly less relaxation in vessels from WHHL rabbits than in vessels from NZW rabbits. Relaxation in response to acetylcholine was greater in carotid arteries from both NZW and WHHL rabbits that were transfected with AdeNOS than in vessels treated with vehicle or Adβgal. Vasorelaxation in response to acetylcholine was inhibited by N^ω-nitro-l-arginine. Responses to sodium nitroprusside were similar after treatment with vehicle alone, Adβgal, or AdeNOS in both groups of rabbits. Thus, overexpression of eNOS with an adenoviral vector improves impaired NO-mediated relaxation in atherosclerotic arteries.