Endothelial cell loss is a critical event in the pathological repair of the injured blood vessel. Impaired endothelial function results in reduced production of key vascular mediators such as nitric oxide (NO) within the vessel wall leading to enhanced smooth muscle cell proliferation and migration and ultimately intimal hyperplasia. The aim of the present study was to directly compare the effects of adenoviral-mediated gene delivery of two nitric oxide synthase (NOS) isoforms, eNOS and iNOS on endothelial regeneration and intimal hyperplasia following endothelial injury in the rabbit carotid artery. The right carotid arteries of male New Zealand white rabbits were denuded by passing a 3French Fogarty balloon catheter along the artery three times. In all, 1 x 10(9) PFU of adenoviral(Ad)eNOS, AdiNOS or Adbeta-galactosidase (Adbeta-Gal) was then delivered intraluminally and allowed to dwell for 20 min. Transgene expression was sought after 3 days by immunohistochemistry and at 7 days by quantitative reverse transcriptase PCR. The effect on intimal hyperplasia was sought using histological staining after 14 days. Evans blue staining was used to determine the effect on endothelial regeneration. eNOS and iNOS expression was detected in transduced arteries. Neointima/media ratios were significantly reduced in eNOS (0.07+/-0.044) and iNOS (0.087+/-0.086) transduced arteries compared with Adbeta-Gal (0.332+/-0.14) transduced arteries (n=7). In addition, AdeNOS treatment (4.21+/-3.12% de-endothelialized area) enhanced endothelial regeneration compared to Adbeta-Gal treatment (10.05+/-4.98), while treatment with AdiNOS (25.17+/-11.92) inhibited endothelial regeneration in the injured rabbit carotid artery (n=7-8). These results highlight the potential of NOS gene therapy, in particular, eNOS gene therapy as a potential therapeutic strategy for the prevention of restenosis after vascular injury.