Background: Adenoviral-mediated gene transfection provides a tool for insertion of foreign genes into non-proliferating cells and tissues in vivo and in vitro. Adenoviral vectors have been used to express recombinant genes in peripheral and cerebral vessels from adult animals of several species. Objective: To determine whether a replication-deficient recombinant adenovirus vector encoding the E. coli β-galactosidase (AdCMVLacZ) reporter gene, driven by the cytomegalovirus promoter, could be used for ex-vivo gene transfer in pulmonary conduit and resistance vessels from newborn piglets.Methods: Viral preparations of the AdCMVLacZ vector grown in monolayers of 293 cells were purified by double cesium gradient ultracentrifugation and dialyzed for 4 hours. Viral titers were determined by plaque assay. Isolated arteries, veins and resistance vessels from newborn piglet lungs were incubated with 2.3 × 108 pfu/mL AdCMVLacZ for 30 minutes followed by incubation in fresh DMEM for 24 hours at 37°C. Vessels were fixed for 30 minutes in 2% paraformaldehyde/0.2% glutaraldehyde and stained with X-Gal reagent for 4 hours. Stained vessels were embedded in paraffin. Serial 4-μm sections were lightly counterstained with eosin for localization to a specific layer of the vessel wall. Results: Transfection with AdCMVLacZ resulted in intense blue staining indicative of expression of recombinant LacZ protein in all vessels studied. No blue staining was observed in non-transfected control vessels fixed and similarly stained. Expression was localized to the adventitia and endothelial cell (EC) layers. Histochemical staining for LacZ protein was not detected in the vascular smooth muscle (VSM) layer of conduit or resistance vessels. This is consistent with our findings of nearly 100% efficiency of LacZ gene transfer in cultured pulmonary microvascular EC and absence of staining in similarly exposed porcine pulmonary VSM cells. Functional studies of vasoreactivity in transfected pulmonary arteries revealed intact VSM and EC responses to U46619 and bradykinin, respectively. Conclusion: These studies demonstrate the successful transfer of recombinant LacZ gene in newborn pulmonary conduit and resistance vessels ex vivo and set the stage for future studies of gene transfer and functional expression using adenoviral vectors encoding other genes, including the gene for human endothelial nitric oxide synthase(AdCMVeNOS).