Fetal gene therapy offers the promise of cure for certain genetic diseases, like cystic fibrosis and surfactant protein B deficiency. The authors hypothesized that a fetoscopic approach could attain a high level of organ-specific gene transfer to the fetal lung late in gestation. To test this hypothesis the authors examined the efficacy, specificity, and toxicity of recombinant adenovirus-mediated transfer of the beta-galactosidase marker gene to the lung of late gestation fetal sheep using a fetoscopic technique. Twelve fetal sheep of 125 to 135 days' gestation (term, 145 days) underwent fetoscopic bronchoscopy and intratracheal administration of a replication-deficient adenoviral vector that transduces the beta-galactosidase marker gene. Escape of administered virus was prevented by the fetoscopic deployment of a detachable silicone balloon in the fetal trachea. All fetuses survived until being killed at 2 days after vector delivery for the histopathologic assessment of vector efficacy and specificity. Optimal beta-galactosidase transgene expression was observed at a viral titer of 2 x 10(12) particles per milliliter of administered volume. Expression was greatest in the distal pulmonary parenchyma, particularly in type II pneumocytes, and extended out to the pleura. There was no evidence of gene transfer in either the large conducting airways or in any other fetal organ. The authors have developed a minimally invasive technique for the specific pulmonary delivery of gene therapy vectors to the fetus with no associated acute toxicity. Gene transfer to the late gestation fetus for the treatment of congenital pulmonary disease may be feasible through fetoscopy.