Background: Nitric oxide (NO) has been shown to decrease myointimal hyperplasia in injured blood vessels. We hypothesize inducible NO synthase (iNOS) gene transfer even at low efficiency will provide adequate local NO production to achieve this goal. Materials and Methods: A retroviral vector containing the human iNOS cDNA (DFGiNOS) was used to transfer the iNOS gene into vascular cells and isolated blood vessels to answer the following questions: can vascular endothelial and smooth muscle cells support iNOS activity and will low efficiency iNOS gene transfer suppress myointimal hyperplasia in injured porcine arteries? Results: DFGiNOS-infected sheep pulmonary artery endothelial cells (SPAEC) expressed significant iNOS mRNA and protein, releasing nitrite levels of 155.0 ± 10.7 nmol/mg protein/24 h vs. 5.5 ± 1.1 by control cells. Transduced rat smooth muscle cells (RSMC) also expressed abundant iNOS mRNA and protein, but, in contrast to SPAEC, NO synthesis was dependent on exogenous tetrahydrobiopterin (BH4) (291.8 ± 10.4 nmol nitrite/mg protein/24 hr with BH4, 37.7 ± 2.6 without BH4). Only porcine arteries infected with DFGiNOS following balloon injury exhibited a 3-fold increase in total NO synthesis and a 15-fold increase in cGMP levels over control vessels in a BH4 dependent fashion, despite only a 1% gene transfer efficiency. Transfer of iNOS completely prevented the 53% increase in myointimal thickness induced by balloon catheter injury; the administration of a NOS inhibitor reversed this effect. Conclusions: These in vitro findings suggest that vascular iNOS gene transfer may be feasible. Furthermore, a low gene transfer efficiency may be sufficient to inhibit myointimal hyperplasia following arterial balloon injury, although a source of BH4 may be required.