Many human genetic diseases, such as congenital surfactant protein B deficiency, manifest in the perinatal period. Prenatal gene therapy may be necessary to minimize morbidity in these diseases. We hypothesized that bacterial beta-galactosidase (beta-Gal) gene could be transferred to and expressed in the pulmonary epithelium of fetal sheep in utero using a replication-deficient adenovirus (Av1LacZ4). We instilled Av1LacZ4 (1.5 x 10(11) plaque-forming units, n = 10) or saline (n = 2) intratracheally to chronically instrumented fetal sheep at 112-134 days gestation (term = 145 days). Lung fluid was collected before and after Av1LacZ4 administration for cytological analysis. Lung tissue was examined for transgenic beta-Gal activity and evidence of toxicity. Transgenic beta-Gal activity was visualized as blue nuclear staining of tissue treated with X-Gal and was detected in the lungs of 5 animals for up to 14 days after administration. Transgenic beta-Gal activity was not detected in the lungs of animals analyzed beyond 14 days after treatment. Pulmonary histopathology was detected in most Av1LacZ4-treated animals and manifested as a mixed cellular infiltrate consisting of neutrophils, macrophages, and lymphocytes. Fetal lung fluid analysis revealed a predominantly lymphocytic response in most Av1LacZ4-treated animals within 3 days (2.88 x 10(6) vs. 4 x 10(3) total cells/ml in control animals). We have demonstrated that adenovirus vectors can direct gene transfer to the lungs of fetal sheep in utero. The transferred gene expression was transient and possibly limited by the induced inflammatory response.