The effects of in vivo gene transfer of endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) genes on severe atherosclerosis were investigated in rabbits. The recombinant adenoviruses, Ad.eNOS and Ad.iNOS, which respectively express eNOS and iNOS, were constructed. Atherosclerosis was induced by a balloon injury followed by a high cholesterol diet for 12 weeks. The rabbits were divided into six groups: Gp cont (no treatment); Gp null (adenovirus sham-infected); Gp eNOS (Ad.eNOS); Gp iNOS (Ad.iNOS); Gp e+i (Ad.eNOS plus Ad.iNOS); and Gp heNOS (a high dose of Ad.eNOS). Examinations were carried out 7 days after gene transfer. Plasma lipid levels were not significantly changed, but transfection with Ad.eNOS (Gp eNOS and Gp heNOS) decreased the tissue cholesterol concentration and regressed atherosclerotic lesions. Vessels treated with Ad.iNOS (Gp iNOS and Gp e+i) showed iNOS staining in the atheroma, and slight staining at other parts of the vessels; those treated with Ad.eNOS showed eNOS staining in the endothelium and subintima, and slight staining at other parts. Ad.eNOS transfection, but not Ad.iNOS or Ad.eNOS+Ad.iNOS transfection, improved the impaired aortic endothelium-dependent relaxation (EDR) and basal NO-dependent response, increased tissue cyclic GMP (cGMP), and decreased the release of O2- from vessels. eNOS treatment showed a decreasing tendency in regions with peroxynitrite staining, MMP1 staining, and suspected apoptosis. In conclusion, in vivo gene transfer of eNOS, but not iNOS or eNOS plus iNOS, regressed atherosclerosis. The relations among NO, O2-, and peroxynitrite may be critical, and lipid resorption from the lesions may be responsible for the regression.