In early gestation the internal surface of the lung is structurally simple and an ideal target for somatic gene transfer. The transfer of genes into the growing lung would be particularly useful in the prenatal correction of cystic fibrosis, which has devastating pulmonary complications. In addition, in utero gene therapy has the potential to immunotolerize the individual, and thereby to avoid the immune reactions now seen with the current generation of adenoviral vectors. We injected a replication-defective adenoviral vector containing the lacZ reporter gene (Ad5.CMVlacZ) into the amniotic fluid of rat pups on the 16th day of gestation. At 16 days of gestation, rat lungs are equivalent in maturity to those of a 22-week human fetus as their airways are lined with undifferentiated multipotential stem cells. The pups showed high-level reporter gene expression in their airways a week following birth (13 days following infection). The expression was maintained during a time when the lung volume increased approximately 20-fold, alveolarization occurred, and the epithelial cells differentiated. These data establish gene targeting of undifferentiated fetal cells as an effective means of gene therapy.