Cystic fibrosis is a common lethal genetic disease caused by functional absence of the cystic fibrosis transmembrane conductance regulator (CFTR). Although a candidate disease for in utero gene therapy, demonstration of potentially therapeutic levels of transgene expression in the fetal airways after minimally invasive gene delivery is a mandatory prerequisite before application of this approach in humans can be considered. We report here on the delivery of a beta-galactosidase expressing adenovirus directly to the airways of fetal sheep in utero using ultrasound-guided percutaneous injection of the trachea in the fetal chest. Injection of adenoviral particles to the fetal airways was not associated with mortality and resulted in low-level expression in the peripheral airways. However, complexation of the virus with DEAE dextran, which confers a positive charge to the virus, and pretreatment of the airways with Na-caprate, which opens tight junctions, increased transgene expression, and a combination of these two enhancers resulted in widespread and efficient gene transfer of the fetal trachea and bronchial tree. Using a percutaneous ultrasound-guided injection technique, we have clearly demonstrated proof of principle for substantial transgene delivery to the fetal airways providing levels of gene expression that could be relevant for a therapeutic application of CFTR expressing vectors.